Biology subjects

Tranchant, E. E.

Publications and source records attributed to Tranchant, E. E..

3 recordsLinked to original sources

bioRxiv2025

Integrative structural analysis of the human endosomal Na⁺/H⁺ exchanger NHE6 reveals a lipid-stabilized intracellular gate and a disordered C-terminus

Human NHE6 (HsNHE6) is an endosomal Na+/H+ exchanger essential for maintaining luminal pH and endo-lysosomal trafficking in neurons. HsNHE6 mutations are implicated in devastating neurological syndromes, but mechanistically the transporter remains poorly understood. Here, we present the single particle cryo-electron microscopy structure of HsNHE6 at 3.4 [A], captured in an inward-open conformation. The structure reveals a homodimeric architecture with 13 transmembrane helices per protomer and the conserved ion-binding site. Functional assays demonstrate that HsNHE6 mediates the exchange of both Na+ and K+ for protons. A structured C-terminal helix interacts with the transmembrane core, jointly forming a hydrophobic cavity that accommodates two lipid molecules, potentially modulating cation access to the ion-binding site. The distal C-terminus of HsNHE6 is intrinsically disordered, as revealed by NMR and small-angle X-ray scattering and equips HsNHE6 with a large cytosolic span. Our integrative model of full-length HsNHE6 provides a comprehensive framework for understanding HsNHE6-mediated ion exchange and its disruption in neurological diseases such as Christianson syndrome.

biochemistry↗

bioRxiv2023

Revisiting the use of dioxane as a reference compound for determination of the hydrodynamic radius of proteins by pulsed field gradient NMR spectroscopy

Measuring the compaction of a protein or complex is key to understand the interactions within and between biomolecules. Experimentally, protein compaction is often probed either by estimating the radius of gyration (Rg) obtained from small-angle X-ray scattering (SAXS) experiments or the hydrodynamic radius (Rh) obtained for example by pulsed field gradient nuclear magnetic resonance (PFG NMR) spectroscopy. PFG NMR experiments generally report on the translational diffusion coefficient, which in turn can be used to estimate Rh using an internal standard. Here, we examine the use of 1,4-dioxane as an internal NMR standard to account for sample viscosity and uncertainty about the gradient strength. Specifically, we revisit the basis for the commonly used reference value for the Rh of dioxane (2.12 [A]) that is used to convert measured diffusion coefficients into a hydrodynamic radius. We follow the same approach that was used to establish the current reference value for the Rh by measuring SAXS and PFG NMR data for a set of seven different proteins and using these as standards. Our analysis shows that the current Rh reference value for 1,4-dioxane Rh (2.12 [A]) is underestimated, and we instead suggest a new value of 2.27 [A] {+/-} 0.04 [A]. Using this updated reference value results in a [~]7% increase in Rh values for proteins whose hydrodynamic radius have been measured by PFG NMR. We discuss the implications for ensemble descriptions of intrinsically disordered proteins and evaluation of effect resulting from for example ligand binding, posttranslational modifications, or changes to the environment.

biophysics↗

bioRxiv2022

Assessment of models for calculating the hydrodynamic radius of intrinsically disordered proteins

Diffusion measurements by pulsed field gradient NMR and fluorescence correlation spectroscopy can be used to probe the hydrodynamic radius of proteins, which contains information about the overall dimension of a protein in solution. The comparison of this value with structural models of intrinsically disordered proteins is nonetheless impaired by the uncertainty of the accuracy of the methods for computing the hydrodynamic radius from atomic coordinates. To tackle this issue, we here build conformational ensembles of 11 intrinsically disordered proteins that we ensure are in agreement with measurements of compaction by small-angle X-ray scattering. We then use these ensembles to identify the forward model that more closely fits the radii derived from pulsed field gradient NMR diffusion experiments. Of the models we examined, we find that the Kirkwood-Riseman equation provides the best description of the hydrodynamic radius probed by pulsed field gradient NMR experiments. While some minor discrepancies remain, our results enable better use of measurements of the hydrodynamic radius in integrative modelling and for force field benchmarking and parameterization. SIGNIFICANCEAccurate models of the conformational properties of intrinsically disordered proteins rely on our ability to interpret experimental data that reports on the conformational ensembles of these proteins in solution. Methods to calculate experimental observables from conformational ensembles are central to link experiments and computation, for example in integrative modelling or the assessment of molecular force fields. Benchmarking such methods is, however, difficult for disordered proteins because it is difficult to construct accurate ensembles without using the data. We here circumvent this problem by combining independent measures of protein compaction to test several methods to calculate the hydrodynamic radius of a disordered protein, as measured by pulsed field gradient NMR diffusion experiments, and find the Kirkwood-Riseman model to be most accurate.

biophysics↗