The Bateman domain of IMP dehydrogenase is a binding target for dinucleoside polyphosphates

Abstract

IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the rate-limiting step in the de novo guanine nucleotide biosynthetic pathway. Because of its involvement in the control of cell division and proliferation, IMPDH represents a therapeutic for managing several diseases, including microbial infections and cancer. IMPDH must be tightly regulated, but the molecular mechanisms responsible for its physiological regulation remain unknown. To this end, we recently reported an important role of adenine and guanine mononucleotides that bind to the regulatory Bateman domain to allosterically modulate the catalytic activity of eukaryotic IMPDHs. Here, we have used enzyme kinetics, X-ray crystallography, and small-angle X-ray scattering (SAXS) methodologies to demonstrate that adenine/guanine dinucleoside polyphosphates bind to the Bateman domain of IMPDH from the fungus Ashbya gossypii with submicromolar affinities. We found that these dinucleoside polyphosphates modulate the catalytic activity of IMPDHs in vitro by efficiently competing with the adenine/guanine mononucleotides for the allosteric sites. These results suggest that dinucleoside polyphosphates play important physiological roles in the allosteric regulation of IMPDHs by adding an additional mechanism for fine-tuning the activities of these enzymes. We propose that these findings may have important implications for the design of therapeutic strategies to inhibit IMPDHs.

Keywords: Bateman domain; IMP dehydrogenase; X-ray crystallography; allosteric regulation; conformational change; conformational switch; dinucleoside polyphosphates; molecular sensor; small-angle X-ray scattering (SAXS); structural biology.

Figure 1.

Nucleotide binding to the Bateman domain of IMDPH. A, cartoon representation of the structure of AgIMPDH octamers formed in the presence of GDP (orange sticks). The catalytic domain is shown in light gray, and the Bateman regulatory domain is colored in blue. The right panel corresponds to a close-up view of two interacting Bateman domains within the octamer, showing the disposition of the three bound GDP molecules. The approximate position of the symmetry axis is indicated. B, close-up views of the adenine and guanine nucleotides (shown in sticks) bound to the two canonical sites of AgIMPDH (represented in semitransparent blue cartoons). Upper left panel, AgIMPDH–ATP (PDB code 5MCP). Upper right panel, HsIMPDH–GTP (PDB code 6I0O). Lower left panel, AgIMPDH–GDP (PDB code 4Z87). Lower right panel, AgIMPDH–ATP/GDP (PDB code 5TC3). In all cases, it can be observed that the β- and γ-phosphates of the nucleotides bound to the canonical sites face each other. The canonical binding sites (1 and 2) are indicated after the corresponding nucleotides; i.e. ATP1 means ATP bound to the canonical site 1.

Figure 2.

Effects of dinucleoside polyphosphates on the catalytic activity of AgIMPDH in vitro. Graphs show the normalized V_max values (V_max^app in the presence of nucleotide divided by the V_max in the absence of nucleotide) versus nucleotide concentration. A and C show the effects of the indicated dinucleoside polyphosphates alone on the AgIMPDH enzyme, whereas B and D show their effects on the enzyme in the presence of GDP (B, inhibited enzyme at 3 mm GDP (21); D, subsaturating GDP concentration of 0.3 mm, which cannot inhibit the enzyme significantly (21)). Experimentally determined V_max values were fitted to a dose-response function (four parameters, variable slope) as implemented in GraphPad Prism software (continuous lines). Error bars represent S.E.

Figure 3.

Structure of AgIMPDH bound to dinucleoside polyphosphates. A, crystal structure of the ternary complex AgIMPDH–Ap5G–GDP. Shown is a close-up view of Ap5G (shown in sticks) simultaneously bound to the two canonical sites of the Bateman domain. The protein is represented in semitransparent blue cartoons with the side chain of key interacting residues shown in sticks. The adjacent monomer and the side chain of residue Arg-167′ are shown in green cartoon and sticks, respectively. The gray mesh around Ap5G represents the 2mF_o − DF_c electron density map contoured at the 1.3σ level (a stereoview of the final F_o − F_c omit map is shown in Fig. S2A). Key protein–nucleotide atomic interactions are represented as yellow dashed lines. B, ATP and GDP mononucleotide structures bound to AgIMPDH (PDB code 5TC3 (21)) compared with the bound structure of Ap5G (PDB code 6RPU, this work). The structural alignment was generated by superimposing the backbone atoms of the Bateman domains (residues 130–210) of both structures. C, SAXS profiles of AgIMPDH in the presence of different mononucleotides and dinucleoside polyphosphates. To facilitate visualization, the plots have been conveniently displaced along the y axis and grouped according to three different conformations: tetramers, extended, and compacted octamers. The profiles shown correspond to: (i) tetramers: 1, control; 2, 0.3 mm GDP; (ii) extended octamers: 3, 3 mm ATP; 4, 0.1 mm Ap6A; 5, 0.1 mm Ap6A + 3 mm GDP; (iii) compacted octamers: 6, 3 mm GDP; 7, 0.1 mm Ap5G + 0.3 mm GDP; 8, 0.1 mm Ap4A + 3 mm GDP; 9, 3 mm ATP + 3 mm GDP.

Figure 4.

Effects of dinucleoside polyphosphates on the catalytic activity of HsIMPDH1 in vitro. Scatter plots show the normalized V_max values (V_max^app in the presence of nucleotide divided by the V_max in the absence of nucleotide) of several adenine dinucleoside polyphosphates compared with ATP (A) as well as Ap5G and Gp5G alone and in combination with GDP at a subsaturating concentration of 0.3 mm (B). V_max values are derived from the Michaelis–Menten analysis of the experimental data. The empty symbols (light gray) are the values obtained from independent experiments, and the error bars (black) represent S.E.