Biology subjects

Harding, B. D.

Publications and source records attributed to Harding, B. D..

4 recordsLinked to original sources

bioRxiv2026

Protonated Structure of EmrE Reveals C-terminal Tail Gating Mechanism

The multidrug efflux pump EmrE is one of the smallest known active transporters and has become a model system for studying multidrug recognition and transport. While recent high-resolution structures have illuminated its dynamic substrate binding pocket, the conformations of its interhelical loops and C-terminal tail, regions critical for controlling proton coupling and gating, remain poorly characterized. Here, we report the high-resolution structure of protonated S64V EmrE determined using solution and solid-state NMR data. This new structural model shows the C-terminal tail occluding the open face of the transport pore, providing a structural basis for how EmrE minimizes proton leak in the absence of substrate. These findings support growing evidence that relatively simple model transporters must leverage an occluded state during alternating access to avoid physiologically unfavorable proton leak.

biophysics↗

bioRxiv2026

In vitro-prepared A30P alpha-synuclein fibrils adopt the conserved and disease-relevant Greek key fold

The pathological hallmark of Parkinson Disease (PD) is the formation of the protein alpha-synuclein (Asyn) into {beta}-sheet rich, self-templating fibrils in the brain. Since the first atomic structure of wild-type Asyn fibrils was determined nearly a decade ago, several other in vitro structures of hereditary mutant fibrils and structures derived from post-mortem diseased patient tissue have been determined by solid-state nuclear magnetic resonance (SSNMR) spectroscopy and cryo-electron microscopy. These structures have not only expanded the library of structures available for computational modeling of drug binding and therapeutics development but have also given unprecedented insight into the disease specificity and structural polymorphism of Asyn fibrils. Here, we report the high-resolution SSNMR structure of the A30P hereditary mutant Asyn fibril, associated with early-onset PD. Our structural model is calculated using several thousand distance restraints derived from one sample, primarily sourced through 3D 13C-13C-13C correlation experiments. The structure adopts a Greek key topology yet does not include the P30 mutation site within the fibril core. We also introduce a comprehensive method for the rapid comparison of SSNMR spectra between Asyn polymorphs of known structure and validate the A30P fold. Lastly, we find that the structure is highly similar to many other experimental structures of both in vitro and ex vivo Asyn fibrils, including those with other hereditary point mutations, suggesting a conserved accessible fold.

biophysics↗

bioRxiv2025

Top-Down Scoring of Spectral Fitness by Image Analysis for Protein Structure Validation

Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for protein structure determination, but traditional approaches require extensive manual assignment of hundreds to thousands of resonances. Here we present NMRFAM-BPHON, a novel "top-down" approach that treats experimental NMR spectra as continuous grayscale images and quantitatively scores the agreement with simulated spectra generated from candidate protein structures. This method does not require complete resonance assignments, though it can incorporate experimental chemical shifts when available to improve performance. The simulated spectra are generated from postulated resonance assignments, which can be derived either from empirical database predictions, direct interpretation, or a hybrid combination. BPHON employs a physics-based approximate polarization transfer model to predict cross-peak intensities from the internuclear distances in the decoy structure, and models the peak lineshapes using empirical, bulk T2 relaxation rates and literature values for scalar couplings. The resulting simulated spectra are scored relative to the experimental data by normalized cross correlation, yielding a fitness score between 0 and 1. We demonstrate BPHONs ability to discriminate structural models, particularly in the case of 13C-detected magic angle spinning solid-state NMR spectra. The software is packaged with a user-friendly graphical user interface for ChimeraX, enabling advanced NMR analysis accessible without requiring extensive manual analysis.

biophysics↗

bioRxiv2025

Backbone and Sidechain 1H, 15N and 13C Resonance Assignments of a Multidrug Efflux Membrane Protein using Solution and Solid-State NMR

EmrE is a bacterial membrane-embedded multidrug transporter that functions as an asymmetric homodimer. EmrE is implicated in antibiotic resistance, but is now known to confer either resistance or susceptibility depending on the identity of the small molecule substrate. Here, we report both solution- and solid-state NMR assignments of S64V-EmrE at pH 5.8, below the pKa of critical residues E14 and H110. This includes 1H, 15N, and 13C resonance assignments of the backbone, methyl groups (isoleucine, leucine, valine, threonine and alanine) from solution NMR experiments in bicelles, and backbone and side-chain assignments from solid-state NMR 13C-detected experiments in liposomes.

biophysics↗