Cryo-EM structures demonstrate human IMPDH2 filament assembly tunes allosteric regulation.

Matthew C Johnson,Justin M Kollman

doi:10.7554/elife.53243

Abstract

Inosine monophosphate dehydrogenase (IMPDH) mediates the first committed step in guanine nucleotide biosynthesis and plays important roles in cellular proliferation and the immune response. IMPDH reversibly polymerizes in cells and tissues in response to changes in metabolic demand. Self-assembly of metabolic enzymes is increasingly recognized as a general mechanism for regulating activity, typically by stabilizing specific conformations of an enzyme, but the regulatory role of IMPDH filaments has remained unclear. Here, we report a series of human IMPDH2 cryo-EM structures in both active and inactive conformations. The structures define the mechanism of filament assembly, and reveal how filament-dependent allosteric regulation of IMPDH2 makes the enzyme less sensitive to feedback inhibition, explaining why assembly occurs under physiological conditions that require expansion of guanine nucleotide pools. Tuning sensitivity to an allosteric inhibitor distinguishes IMPDH from other metabolic filaments, and highlights the diversity of regulatory outcomes that can emerge from self-assembly.

Highlights

Ribonucleotides play a central role in cellular physiology, and complex regulatory networks maintain optimal nucleotide levels according to the variable metabolic state of the cell (Lane and Fan, 2015)
While the turnover rate we observe here (~0.05 per second) is somewhat low, these reactions were performed near room temperature (24 ̊C); we found that increasing reaction temperature to 37 ̊C resulted in increasing Kcat to ~0.1 per second, roughly half what was observed in another recently published study of human IMPDH2 (Figure 2—figure supplement 2) (Fernandez-Justel et al, 2019)
inosine monophosphate (IMP) dehydrogenase (IMPDH) plays a important role in the immune response, where T-cell activation is dependant on increased production of purine nucleotides, and is associated with IMPDH filament assembly (Calise et al, 2018; Duong-Ly et al, 2018; Gu et al, 2000; Zimmermann et al, 1998)

Summary

Introduction

Ribonucleotides play a central role in cellular physiology, and complex regulatory networks maintain optimal nucleotide levels according to the variable metabolic state of the cell (Lane and Fan, 2015). Structures of the enzyme treated with multiple combinations of substrates (IMP, NAD+) and allosteric effectors (ATP, GTP), in both filament and non-filament assembly states, demonstrate the extreme conformational plasticity of the enzyme These structures define the interactions that drive filament assembly, and we show that in vitro IMPDH2 filament assembly is sensitive to the same conditions that promote assembly in cells: high IMP levels and low guanine nucleotide levels (Keppeke et al, 2018). In other enzyme polymers with known functions, assembly directly up or down regulates activity; the resistance to feedback inhibition observed in IMPDH2 filaments demonstrates a new role for enzyme self-assembly and reflects the diversity of allosteric regulatory mechanisms in metabolic filaments From these data, we have developed a model (described in more detail below), in which IMPDH2 filament assembly modulates known conformational changes of the enzyme to alter catalytic flux in response to proliferative signaling. This allows for a third regulatory state in which the enzyme is able to resist feedback inhibition in response to proliferative signaling

Results

Discussion

Materials and methods

Funding Funder National Institutes of Health