Mechanosensitive ion channels MSL8, MSL9, and MSL10 have environmentally sensitive intrinsically disordered regions with distinct biophysical characteristics in vitro

Aidan J Flynn; Kari Miller; Jennette M Codjoe; Matthew R King; Elizabeth S Haswell

doi:10.1002/pld3.515

Mechanosensitive ion channels MSL8, MSL9, and MSL10 have environmentally sensitive intrinsically disordered regions with distinct biophysical characteristics in vitro

Plant Direct. 2023 Aug 3;7(8):e515. doi: 10.1002/pld3.515. eCollection 2023 Aug.

Authors

Aidan J Flynn^{1

2

3}, Kari Miller^{1

2}, Jennette M Codjoe^{1

2}, Matthew R King⁴, Elizabeth S Haswell^{1

2}

Affiliations

¹ Department of Biology Washington University in St. Louis St. Louis Missouri USA.
² NSF Center for Engineering Mechanobiology, Department of Biology Washington University in St. Louis St. Louis Missouri USA.
³ Department of Biochemistry and Biophysics Washington University in St. Louis St. Louis Missouri USA.
⁴ Department of Biomedical Engineering Washington University in St. Louis St. Louis Missouri USA.

Abstract

Intrinsically disordered protein regions (IDRs) are highly dynamic sequences that rapidly sample a collection of conformations over time. In the past several decades, IDRs have emerged as a major component of many proteomes, comprising ~30% of all eukaryotic protein sequences. Proteins with IDRs function in a wide range of biological pathways and are notably enriched in signaling cascades that respond to environmental stresses. Here, we identify and characterize intrinsic disorder in the soluble cytoplasmic N-terminal domains of MSL8, MSL9, and MSL10, three members of the MscS-like (MSL) family of mechanosensitive ion channels. In plants, MSL channels are proposed to mediate cell and organelle osmotic homeostasis. Bioinformatic tools unanimously predicted that the cytosolic N-termini of MSL channels are intrinsically disordered. We examined the N-terminus of MSL10 (MSL10^N) as an exemplar of these IDRs and circular dichroism spectroscopy confirms its disorder. MSL10^N adopted a predominately helical structure when exposed to the helix-inducing compound trifluoroethanol (TFE). Furthermore, in the presence of molecular crowding agents, MSL10^N underwent structural changes and exhibited alterations to its homotypic interaction favorability. Lastly, interrogations of collective behavior via in vitro imaging of condensates indicated that MSL8^N, MSL9^N, and MSL10^N have sharply differing propensities for self-assembly into condensates, both inherently and in response to salt, temperature, and molecular crowding. Taken together, these data establish the N-termini of MSL channels as intrinsically disordered regions with distinct biophysical properties and the potential to respond uniquely to changes in their physiochemical environment.

Keywords: Arabidopsis thaliana; circular dichroism; intrinsically disordered protein; ion channel; mechanobiology; phase separation; transmembrane protein.