NMR for Biomolecule Conformations and Drug Discovery in Neurodegenerative Diseases
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Nuclear magnetic resonance (NMR) is a uniquely powerful method to interrogate biomolecules at high resolution because it enables scientists to study proteins, oligonucleotides and ribonucleic acid (RNA) under physiological conditions — and inside cells. Proteins, with their unparalleled diversity and complexity, offer an intriguing testing ground for NMR technology. The three-dimensional structure of biomolecules determines their physiological function, and changes in the structure and the formation of abnormal protein conformations lead to disease. Science has benefited from the ability of NMR to shed light on the conformations of biomolecules and the modes of action of small molecules with potential use as drugs.
NMR to determine biomolecular structure
Researchers are using NMR as a method for determining molecular structures and interactions in detail, with the long-term goal of developing potential drugs to treat disease. A prominent example of this work was determining the high-resolution structure of the mammalian translocator protein (TSPO), which has increased expression in areas of brain injury and inflammation. The research demonstrated that coupling TSPO to a diagnostic ligand called PK11195 stabilized its structure, allowing the structure to be determined as a bundle of five helices.1
The 18-kilodalton translocator protein TSPO is found in mitochondrial membranes and mediates the import of cholesterol and porphyrins into mitochondria. In line with the role of TSPO in mitochondrial function, TSPO ligands are used for a variety of diagnostic and therapeutic applications in animals and humans. NMR was used to determine the three-dimensional high-resolution structure of mammalian TSPO reconstituted in detergent micelles in complex with its high-affinity ligand PK11195. The TSPO-PK11195 structure is described by a tight bundle of five transmembrane α helices that form a hydrophobic pocket accepting PK11195. Ligand-induced stabilization of the structure of TSPO suggests a molecular mechanism for the stimulation of cholesterol transport into mitochondria.
High field NMR was crucial to this work, by allowing the proximity of specific 1H, 13C and 15N nuclei to be determined using nuclear Overhauser spectroscopy (NOESY). The result was a better understanding of how TSPO recognizes and binds to diagnostic markers and drugs, with clear diagnostic and therapeutic implications.
NMR also has been instrumental for research on the proteins involved in neurodegenerative disorders, with special focus on three polypeptides – tau, amyloid-β and α-synuclein – that form insoluble deposits that are hallmarks of Alzheimer’s disease and Parkinson’s disease. For example, recent work looked at the processes that underlie the formation of insoluble deposits of tau in the brain of patients with Alzheimer’s disease, and the team identified liquid-liquid phase separation as a critical driving factor.2
For many years NMR spectroscopy has been used to understand the structure of tau, as well as its function and mechanisms of action. NMR offered detailed insights into the structural polymorphism of tau, phosphorylation at different sites, the influence of aggregation inhibitors on tau molecular structures, and the fibrous layer of insoluble tau deposits with unprecedented detail. Additionally, data was gathered regarding the interaction of tau with microtubules and actin filaments, as well as the molecular chaperone Hsp90.
Abnormal protein folding in liquid-liquid phase-separated states, soluble oligomers and amyloid fibrils has been associated with the progression of neurodegenerative diseases, and insoluble deposits of tau and α-synuclein proteins are pathological features of various neurodegenerative diseases such as Alzheimer's and Parkinson's. This study provided mechanistic insights into the misfolding and pathogenic association of the protein tau in Alzheimer's disease through biophysical analysis using high-resolution NMR spectroscopy and biochemical experiments as well as collaborative studies in cellular/animal models of Alzheimer's. The goal was to identify new targets/conformations/mechanisms for small-molecule intervention, and, thus, new strategies for the diagnosis and therapy of neurodegenerative diseases.
The molecular crowding of tau that is a consequence of the phase separation process was uncovered by two-dimensional 1H/15N correlation spectroscopy (HSQC) of tau coupled to a paramagnetic tag. The findings in this study triggered a wide range of research in different labs worldwide.
Figure 1: Molecular crowding of the Alzheimer’s disease associated protein tau in liquid-liquid phase separated condensates revealed by NMR spectroscopy. (A) Liquid droplets of tau visualized by fluorescence microscopy. (B) Paramagnetic broadening in 2D 1H-13C HSQC spectra of the microtubule-binding domain of tau at 5°C (left; dispersed phase) and 37°C (right; phase separated conditions) as seen for its four threonine residues. The microtubule-binding domain of tau was tagged with MTSL at its two native cysteines. Paramagnetic and diamagnetic states are represented by gold and black color, respectively. Reprinted from ref.  under a Creative Commons license (CC BY 4.0, http://creativecommons.org/licenses/by/4.0/).
NMR allows scientists to study how the biomolecule moves around in solution, and the different shapes it takes on to perform different activities. Additionally, it enables the visualization of molecules in real-time, gaining crucial insights into how they perform their function and are modified by enzymes. The goal of this pioneering work is to lead to better treatment for those suffering from neurodegenerative diseases.
1. Jaremko Ł, Jaremko M, Giller K, Becker S, Zweckstetter M. Structure of the mitochondrial translocator protein in complex with a diagnostic ligand. Science. 2014;343(6177):1363-1366. doi: 10.1126/science.1248725
2. Ambadipudi S, Biernat J, Riedel D, Mandelkow E, Zweckstetter M. Liquid–liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein Tau. Nat Commun. 2017;8(1):275. doi: 10.1038/s41467-017-00480-0
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