Structural basis of dynamic P5CS filaments

Abstract

The bifunctional enzyme Δ1-pyrroline-5-carboxylate synthase (P5CS) is vital to the synthesis of proline and ornithine, playing an essential role in human health and agriculture. Pathogenic mutations in P5CS gene (ALDH18A1) lead to neurocutaneous syndrome and skin relaxation connective tissue disease in humans, and P5CS deficiency seriously damages the ability to resist adversity in plants. We have recently found that P5CS forms the cytoophidium in vivo and filaments in vitro. However, it is difficult to appreciate the function of P5CS filamentation without precise structures. Using cryo-electron microscopy, here we solve structures of Drosophila full-length P5CS in three states at resolution from 3.1 to 4.3 Å. We observe distinct ligand-binding states and conformational changes for the GK and GPR domains, respectively. Divergent helical filaments are assembled by P5CS tetramers and stabilized by multiple interfaces. Point mutations disturbing those interfaces prevent P5CS filamentation and greatly reduce the enzymatic activity. Our findings reveal that filamentation is crucial for the coordination between the GK and GPR domains, providing structural basis for catalytic function of P5CS filaments.

Dynamic atP5CS2 Filament Facilitates Substrate Channeling

“…Compared with dmP5CS, the regulation of atP5CS2 filament is different. For dmP5CS, the contribution of glutamate is greater than ATP 7 . For atP5CS2, both glutamate and ATP promote filament formation at similar degrees ( Figure 2 C ).…”

“…Some well-studied filamentous enzymes, like CTPS and PRPS, have shown that their filamentation is conserved across a wide range of species [36][37][38][39][40][41] . In previous studies, we found dmP5CS could form cytoophidia and filament, and the filament is critical for its activity 7 . But the conservation of P5CS filament was not clear.…”

“…Some studies have suggested that there are some interactions between prokaryotic GK and GPR, which may have significant implications for substrate channeling of G5P 9,10 . For dmP5CS, mutations that disrupt filamentation can also impair overall activity dramatically 7 . It has been assumed that dmP5CS filaments promote G5P channeling, but the detailed mechanism between filamentation and enzyme activity is still unclear.…”

“…We have found that Drosophila melanogaster P5CS (dmP5CS) can form cytoophidium, and purified dmP5CS can also form filament in vitro 6 . In dmP5CS filaments, GK tetramers are assembled as the “core” of the filaments, while GPR dimers surround GK tetramers in a double helix pattern 7 . The ordered arrangement of GK and GPR in dmP5CS may be encouraged during evolution, as the intermediate product G5P is highly unstable 8 .…”

Light-sensitive phosphorylation regulates enzyme activity and filament assembly of human IMPDH1 retinal splice variants

“…More than 20 different metabolic enzymes form filaments in cells, suggesting that filament assembly is a general mechanism for regulating multiple metabolic pathways (Hvorecny and Kollman, 2023; Lynch et al, 2020; Narayanaswamy et al, 2009; Noree et al, 2010; Park and Horton, 2019; Park and Horton, 2020; Shen et al, 2016; Simonet et al, 2020). For many metabolic enzymes, filament formation is an important mechanism for fine-tuning catalytic activity (Barry et al, 2014; Hansen et al, 2021; Hu et al, 2022; Hunkeler et al, 2018; Hvorecny et al, 2023; Lu et al, 2023; Lynch and Kollman, 2020; Lynch et al, 2017; Polley et al, 2019; Stoddard et al, 2020; Zhong et al, 2022). Here, we demonstrate that filament assembly itself can be directly tuned by phosphorylation, adding an additional layer to an already complex mode of regulation for IMPDH that occurs at different levels.…”