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In-Lipid Structure of Pressure-Sensitive Domains Hints Mechanosensitive Channel Functional Diversity.

ABSTRACT: The mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis has been used as a structural model for rationalizing functional observations in multiple MscL orthologs. Although these orthologs adopt similar structural architectures, they reportedly present significant functional differences. Subtle structural discrepancies on mechanosensitive channel nanopockets are known to affect mechanical gating and may be linked to large variability in tension sensitivity among these membrane channels. Here, we modify the nanopocket regions of MscL from Escherichia coli and M. tuberculosis and employ PELDOR/DEER distance and 3pESEEM deuterium accessibility measurements to interrogate channel structure within lipids, in which both channels adopt a closed conformation. Significant in-lipid structural differences between the two constructs suggest a more compact E. coli MscL at the membrane inner-leaflet, as a consequence of a rotated TM2 helix. Observed differences within lipids could explain E. coli MscL's higher tension sensitivity and should be taken into account in extrapolated models used for MscL gating rationalization.

SUBMITTER: Kapsalis C

PROVIDER: S-EPMC7376121 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Echinocandins show fungicidal activity against common invasive mycoses but are ineffective against cryptococcosis. The underlying mechanism for echinocandin resistance in Cryptococcus neoformans remains poorly understood but has been shown to involve Cdc50, the regulatory subunit of lipid flippase. In a forward genetic screen for cdc50? suppressor mutations that are caspofungin resistant, we identified Crm1 (caspofungin resistant mutation 1), a homolog of mechanosensitive channel proteins, and showed that crm1? restored caspofungin resistance in cdc50? cells. Caspofungin-treated cdc50? cells exhibited abnormally high intracellular calcium levels ([Ca2+]c) and heightened activation of the calcineurin pathway. Deletion of CRM1 in the cdc50? background normalized the abnormally high [Ca2+]c. Cdc50 interacts with Crm1 to maintain cellular calcium homeostasis. Analysis of chitin/chitosan content showed that deleting CRM1 reversed the decreased chitosan production of cdc50? cells. Together, these results demonstrate that Cdc50 and Crm1 regulation of the calcineurin pathway and cytoplasmic calcium homeostasis may underlie caspofungin resistance in C. neoformans IMPORTANCE Cryptococcus neoformans is the leading cause of fungal meningitis, accounting for ?15% of HIV/AIDS-related deaths, but treatment options for cryptococcosis are limited. Echinocandins are the newest fungicidal drug class introduced but are ineffective in treating cryptococcosis. Our previous study identified the lipid flippase subunit Cdc50 as a contributor to echinocandin resistance in C. neoformans Here, we further elucidated the mechanism of Cdc50-mediated caspofungin drug resistance. We discovered that Cdc50 interacts with the mechanosensitive calcium channel protein Crm1 to regulate calcium homeostasis and caspofungin resistance via calcium/calcineurin signaling. These results provide novel insights into echinocandin resistance in this pathogen, which may lead to new treatment options, as well as inform echinocandin resistance mechanisms in other fungal organisms and, hence, advance our understanding of modes of antifungal drug susceptibility and resistance.

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In-Lipid Structure of Pressure-Sensitive Domains Hints Mechanosensitive Channel Functional Diversity.

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