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McGrath, A. P.

Publications and source records attributed to McGrath, A. P..

2 recordsLinked to original sources

Cryo-EM Structure of OSCA1.2 from Oryza sativa: Mechanical basis of hyperosmolality-gating in Plants

Sensing and responding to environmental water deficiency and osmotic stresses is essential for the growth, development and survival of plants. Recently, an osmolality-sensing ion channel called OSCA1 was discovered that functions in sensing hyperosmolality in Arabidopsis. Here, we report the cryo-EM structure and function of an ion channel from rice (Oryza sativa; OsOSCA1.2), showing how it mediates hyperosmolality sensing and ion permeability. The structure reveals a dimer; the molecular architecture of each subunit consists of eleven transmembrane helices and a cytosolic soluble domain that has homology to RNA recognition proteins. The transmembrane domain is structurally related to the TMEM16 family of calcium dependent ion channels and scramblases. The cytosolic soluble domain possesses a distinct structural feature in the form of extended intracellular helical arms that is parallel to the plasma membrane. These helical arms are well positioned to sense lateral tension on the inner leaflet of the lipid bilayer caused by changes in turgor pressure. Computational dynamic analysis suggests how this domain couples to the transmembrane portion of the molecule to open the channel. Hydrogen-deuterium exchange mass spectrometry (HDXMS) experimentally confirms the conformational dynamics of these coupled domains. The structure provides a framework to understand the structural basis of hyperosmolality sensing in an important crop plant, extends our knowledge of the anoctamin superfamily important for plants and fungi, and provides a structural mechanism for translating membrane stress to ion transport regulation.

biophysics↗

Functional characterization and discovery of modulators of SbMATE, the agronomically important aluminium tolerance transporter from Sorghum bicolor

About 50% of the worlds arable land is strongly acidic (soil pH < 5). The low pH of these soils solubilizes root-toxic ionic aluminium (Al3+) species from clay minerals, driving the evolution of various counteractive adaptations in cultivated crops. The food crop Sorghum bicolor, for example, upregulates the membrane-embedded transporter protein SbMATE in its roots. SbMATE mediates efflux of the anionic form of the organic acid, citrate, into the soil rhizosphere, chelating Al3+ ions and thereby imparting Al-resistance based on excluding Al+3 from the growing root tip. Here, we use electrophysiological, radiolabeled, and fluorescence-based transport assays in two heterologous expression systems to establish a broad substrate recognition profile of SbMATE, showing the transport of 14C- citrate anion, as well as the organic monovalent cation, ethidium, but not the divalent ethidium-derivative, propidium. The transport cycle is proton and/or sodium-driven, and shares certain molecular mechanisms with bacterial MATE-family transporters. We further complement our transport assays by directly measuring substrate binding to detergent-purified SbMATE protein. Finally, we use the functionally-folded, purified membrane protein as an antigen to discover high-affinity, native conformation-binding and transport function-altering nanobodies using an animal-free, mRNA/cDNA display technology. Our results demonstrate the utility of using Pichia pastoris as an efficient eukaryotic host to express large quantities of functional plant transporter proteins for in vitro characterization. The nanobody discovery approach is applicable to other low immunogenic plant proteins.

plant biology↗