High-resolution structural information of membrane-bound α-synuclein provides insight into the MoA of the anti-Parkinson drug UCB0599.

α-synuclein (αS) is an intrinsically disordered protein whose functional ambivalence and protein structural plasticity are iconic. Coordinated protein recruitment ensures proper vesicle dynamics at the synaptic cleft, while deregulated oligomerization on cellular membranes contributes to cell damage and Parkinson's disease (PD). Despite the protein's pathophysiological relevance, structural knowledge is limited. Here, we employ NMR spectroscopy and chemical cross-link mass spectrometry on 14N/15N-labeled αS mixtures to provide for the first time high-resolution structural information of the membrane-bound oligomeric state of αS and demonstrate that in this state, αS samples a surprisingly small conformational space. Interestingly, the study locates familial Parkinson's disease mutants at the interface between individual αS monomers and reveals different oligomerization processes depending on whether oligomerization occurs on the same membrane surface (cis) or between αS initially attached to different membrane particles (trans). The explanatory power of the obtained high-resolution structural model is used to help determine the mode-of-actionof UCB0599. Here, it is shown that the ligand changes the ensemble of membrane-bound structures, which helps to explain the success this compound, currently being tested in Parkinson's disease patients in a phase 2 trial, has had in animal models of PD.

Synthesis of a 13C-methylene-labeled isoleucine precursor as a useful tool for studying protein side-chain interactions and dynamics.

In this study, we present the synthesis and incorporation of a metabolic isoleucine precursor compound for selective methylene labeling. The utility of this novel α-ketoacid isotopologue is shown by incorporation into the protein Brd4-BD1, which regulates gene expression by binding to acetylated histones. High quality single quantum 13C-1 H-HSQC were obtained, as well as triple quantum HTQC spectra, which are superior in terms of significantly increased 13C-T2 times. Additionally, large chemical shift perturbations upon ligand binding were observed. Our study thus proves the great sensitivity of this precursor as a reporter for side-chain dynamic studies and for investigations of CH-π interactions in protein-ligand complexes.

13 Cß-Valine and 13 Cγ-Leucine Methine Labeling To Probe Protein Ligand Interaction.

Precise information regarding the interaction between proteins and ligands at molecular resolution is crucial for effectively guiding the optimization process from initial hits to lead compounds in early stages of drug development. In this study, we introduce a novel aliphatic side chain isotope-labeling scheme to directly probe interactions between ligands and aliphatic sidechains using NMR techniques. To demonstrate the applicability of this method, we selected a set of Brd4-BD1 binders and analyzed 1 H chemical shift perturbation resulting from CH-π interaction of Hß -Val and Hγ -Leu as CH donors with corresponding ligand aromatic moieties as π acceptors.

Probing Protein-Ligand Methyl-π Interaction Geometries through Chemical Shift Measurements of Selectively Labeled Methyl Groups.

Fragment-based drug design is heavily dependent on the optimization of initial low-affinity binders. Herein we introduce an approach that uses selective labeling of methyl groups in leucine and isoleucine side chains to directly probe methyl-π contacts, one of the most prominent forms of interaction between proteins and small molecules. Using simple NMR chemical shift perturbation experiments with selected BRD4-BD1 binders, we find good agreement with a commonly used model of the ring-current effect as well as the overall interaction geometries extracted from the Protein Data Bank. By combining both interaction geometries and chemical shift calculations as fit quality criteria, we can position dummy aromatic rings into an AlphaFold model of the protein of interest. The proposed method can therefore provide medicinal chemists with important information about binding geometries of small molecules in fast and iterative matter, even in the absence of high-resolution experimental structures.

On-Cell NMR Contributions to Membrane Receptor Binding Characterization.

NMR spectroscopy has matured into a powerful tool to characterize interactions between biological molecules at atomic resolution, most importantly even under near to native (physiological) conditions. The field of in-cell NMR aims to study proteins and nucleic acids inside living cells. However, cells interrogate their environment and are continuously modulated by external stimuli. Cell signaling processes are often initialized by membrane receptors on the cell surface; therefore, characterizing their interactions at atomic resolution by NMR, hereafter referred as on-cell NMR, can provide valuable mechanistic information. This review aims to summarize recent on-cell NMR tools that give information about the binding site and the affinity of membrane receptors to their ligands together with potential applications to in vivo drug screening systems.