Dhungel, S. | Search bioRxiv

bioRxiv2024

Nutrient Signaling-Dependent Quaternary Structure Remodeling Drives the Catalytic Activation of metazoan PASK

The Per-Arnt-Sim (PAS) domains are characterized by diverse sequences and feature tandemly arranged PAS and PAS-associated C-terminal (PAC) motifs that fold seamlessly to generate the metabolite-sensing PAS domain. Here, using evolutionary scale sequence, domain mapping, and deep learning-based protein structure analysis, we deconstructed the sequence-structure relationship to unearth a novel example of signal-regulated assembly of PAS and PAC subdomains in metazoan PAS domain-regulated kinase (PASK). By comparing protein sequence, domain architecture, and computational protein models between fish, bird, and mammalian PASK orthologs, we propose the existence of previously unrecognized third PAS domain of PASK (PAS-C) formed through long-range intramolecular interactions between the N-terminal PAS fold and the C-terminal PAC fold. We experimentally validated this novel structural design using residue-level cross-linking assays and showed that the PAS-C domain assembly is nutrient-responsive. Furthermore, by combining structural phylogeny approaches with residue-level cross-linking, we revealed that the PAS-C domain assembly links nutrient sensing with quaternary structure reorganization in PASK, stabilizing the kinase catalytic core of PASK. Thus, PAS-C domain assembly likely integrates environmental signals, thereby relaying sensory information for catalytic control of the PASK kinase domain. In conclusion, we theorize that during their horizontal transfer from bacteria to multicellular organisms, PAS domains gained the capacity to integrate environmental signals through dynamic modulation of PAS and PAC motif interaction, adding a new regulatory layer suited for multicellular systems. We propose that metazoan PAS domains are likely to be more dynamic in integrating sensory information than previously considered, and their structural assembly could be targeted by regulatory signals and exploited to develop therapeutic strategies.

biochemistry↗

bioRxiv2023

Signal-regulated unmasking of the nuclear localization motif in the PAS domain regulates the nuclear translocation of PASK.

The ligand-regulated PAS domains are one of the most diverse signal-integrating domains found in proteins from prokaryotes to humans. By biochemically connecting cellular processes with their environment, PAS domains facilitate an appropriate cellular response. PAS domain-containing Kinase (PASK) is an evolutionarily conserved protein kinase that plays important signaling roles in mammalian stem cells to establish stem cell fate. We have shown that the nuclear translocation of PASK is stimulated by differentiation signaling cues in muscle stem cells. However, the mechanistic basis of the regulation of PASK nucleo-cytoplasmic translocation remains unknown. Here, we show that the PAS-A domain of PASK contains a putative monopartite nuclear localization sequence (NLS) motif. This NLS is inhibited in cells via intramolecular association with a short linear motif, termed the PAS Interacting Motif (PIM), found upstream of the kinase domain. The interaction between the PAS-A domain and PIM is evolutionarily conserved and serves to retain PASK in the cytosol in the absence of signaling cues. Consistent with that, we show that metabolic inputs induce PASK nuclear import, likely by disrupting the PAS-A: PIM association. We suggest that a route for such linkage may occur through the PAS-A ligand binding cavity. We show that PIM recruitment and artificial ligand binding to the PAS-A domain occur at neighboring locations that could facilitate metabolic control of the PAS-PIM interaction. Thus, the PAS-A domain of PASK integrates metabolic signaling cues for nuclear translocation and could be targeted to control the balance between self-renewal and differentiation in stem cells.

biochemistry↗