Biology subjects

Stansfeld, P. J.

Publications and source records attributed to Stansfeld, P. J..

39 records · Page 3Linked to original sources

bioRxiv2019

Complete structure of the core signalling unit of the E. coli chemosensory array in an optimised minicell strain

Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays.1,2 Understanding stimulus detection and transmission at the molecular level requires precise structural characterisation of the array building block known as a core signalling unit (CSU). Here we introduce a novel E. coli strain that forms small minicells possessing extended and highly ordered chemosensory arrays. We provide a three-dimensional (3D) map of a complete CSU at ~16 [A] resolution by cryo-electron tomography (cryo-ET) and subtomogram averaging. This map, combined with previously determined high resolution structures and molecular dynamics simulations, yields an atomistic model of the membrane-bound CSU and enables spatial localisation of its signalling domains. Our work thus offers a solid structural basis for interpretation of existing data and design of new experiments to elucidate signalling mechanisms within the CSU and larger array.

cell biology↗

bioRxiv2018

Identification of Hydrophobic Gates by Simulation-Enabled Analysis of the Ion Channel Structural Proteome

Ion channel proteins control ionic flux across biological membranes through conformational changes in their transmembrane pores. An exponentially increasing number of channel structures captured in different conformational states are now being determined. However, these newly-resolved structures are commonly classified as either open or closed based solely on the physical dimensions of their pore and it is now known that more accurate annotation of their conductive state requires an additional assessment of the effect of pore hydrophobicity. A narrow hydrophobic gate region may disfavour liquid-phase water, leading to local de-wetting which will form an energetic barrier to water and ion permeation without steric occlusion of the pore. Here we quantify the combined influence of radius and hydrophobicity on pore de-wetting by applying molecular dynamics simulations and machine learning to nearly 200 ion channel structures. This allows us to propose a simple simulation-free heuristic model that rapidly and accurately predicts the presence of hydrophobic gates. This not only enables the functional annotation of new channel structures as soon as they are determined, but may also facilitate the design of novel nanopores controlled by hydrophobic gates. Significance statementIon channels are nanoscale protein pores in cell membranes. An exponentially increasing number of structures for channels means that computational methods for predicting their functional state are needed. Hydrophobic gates in ion channels result in local de-wetting of pores which functionally closes them to water and ion permeation. We use simulations of water behaviour within nearly 200 different ion channel structures to explore how the radius and hydrophobicity of pores determine their hydration vs. de-wetting behaviour. Machine learning-assisted analysis of these simulations enables us to propose a simple model for this relationship. This allows us to present an easy method for the rapid prediction of the functional state of new channel structures as they emerge.

biophysics↗

bioRxiv2018

The structural basis of lipid scrambling and inactivation in the endoplasmic reticulum scramblase TMEM16K

Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase and ion channel activity, or specific chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident calcium-regulated lipid scramblase. Our crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional structures solved by cryo-EM reveal extensive conformational changes extending from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity. Our results suggest mechanisms by which missense variants of TMEM16K could cause SCAR10 ataxia, providing new hypotheses to explore for therapy.

biochemistry↗