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

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 (MSL10N) as an exemplar of these IDRs and circular dichroism spectroscopy confirms its disorder. MSL10N adopted a predominately helical structure when exposed to the helix‐inducing compound trifluoroethanol (TFE). Furthermore, in the presence of molecular crowding agents, MSL10N underwent structural changes and exhibited alterations to its homotypic interaction favorability. Lastly, interrogations of collective behavior via in vitro imaging of condensates indicated that MSL8N, MSL9N, and MSL10N 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.

Open structure and gating of the Arabidopsis mechanosensitive ion channel MSL10

“…The full-length At MSL10 channel was subjected to structural analysis, but the N-terminal ‘death’ domain (NTD), consisting of amino acids 1-165, could not be resolved in the cryo-EM densities. This is consistent with the finding that the NTD is an intrinsically disordered region (IDR) that interacts with the C-terminal domain of MSL10 to regulate cell death signaling and ion channel activity 43 , 44 . This notion is also reminiscent of the TRPV4 ion channel that is involved in thermo- and osmoregulation, in which the N-terminal IDR modulates channel activity via dynamic long-range interactions 45 .…”