NMR for biomolecular conformations and drug discovery in neurodegenerative diseases

Nuclear magnetic resonance (NMR) is a uniquely powerful method for studying biomolecules with high resolution because it allows 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 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 for biomolecular structure determination

Researchers use MRI as a method to determine molecular structures and interactions in detail, with the long-term goal of developing potential drugs to treat diseases. A striking example of this work was the determination of the high-resolution structure of the mammalian translocator protein (TSPO), which has increased expression in areas of brain injury and inflammation. The study showed that binding TSPO to a diagnostic ligand called PK11195 stabilized its structure, allowing the structure to be defined as a five-helix bundle.1

The 18-kilodalton translocator protein TSPO is located in mitochondrial membranes and mediates the import of cholesterol and porphyrins into mitochondria. Consistent 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 high-resolution three-dimensional structure of mammalian TSPO reconstituted in detergent micelles in complex with its high-affinity ligand PK11195. The structure of TSPO-PK11195 is described by a tight bundle of five transmembrane α helices that form a hydrophobic pocket that accommodates PK11195. Ligand-induced stabilization of the TSPO structure suggests a molecular mechanism for stimulating mitochondrial cholesterol transport.

High-field NMR was critical to this work, 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.

Neurodegenerative disorders

MRI is also a tool for studying proteins involved in neurodegenerative disorders, with a particular focus on three polypeptides—tau, amyloid-β, and α-synuclein—that form the insoluble deposits that are hallmarks of Alzheimer’s disease and Parkinson’s disease. For example, recent work looked at the processes underlying the formation of insoluble tau deposits in the brains of Alzheimer’s patients, and the team identified the 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. MRI offers detailed insight into the structural polymorphism of tau, phosphorylation at different sites, the influence of aggregation inhibitors on the molecular structures of tau, and the fibrous layer of insoluble tau deposits in unprecedented detail. In addition, data were collected on the interaction of tau with microtubules and actin filaments, as well as the molecular chaperone Hsp90.

Breakthrough discovery

Abnormal folding of proteins in liquid–liquid phase-separated states, soluble oligomers and amyloid fibrils is 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 insight into the misfolding and pathogenic association of tau protein 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 aim was to identify new targets/conformations/mechanisms for small molecule intervention and thus new strategies for the diagnosis and therapy of neurodegenerative diseases.

Molecular clustering of tau, which is a consequence of the phase separation process, was detected by two-dimensional 1H/15N correlation spectroscopy (HSQC) of tau bound to a paramagnetic label. The findings in this study sparked a wide range of research in different laboratories around the world.

Figure 1: Molecular clustering 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 MTSL-tagged at its two native cysteines. The paramagnetic and diamagnetic states are represented in gold and black, respectively. Reprinted from ref. [2] under a Creative Commons license (CC BY 4.0, http://creativecommons.org/licenses/by/4.0/).

NMR allows scientists to study how a biomolecule moves in solution and the different shapes it takes to perform different activities. Furthermore, it enables real-time visualization of molecules, gaining crucial insight into how they perform their function and how they are modified by enzymes. The goal of this pioneering work is to lead to better treatments for those suffering from neurodegenerative diseases.

References:

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 Alzheimer’s-associated protein Yes Nat Common. 2017; 8 (1): 275. doi: 10.1038/s41467-017-00480-0

About the author:

Prof. Dr. Markus Zweckstetter is head of the NMR-based protein structure research group at the Max Planck Institute for Multidisciplinary Sciences in Göttingen, Germany. Since 2012, he also heads the Translational Structural Biology Group at the German Center for Neurodegenerative Diseases (DZNE) and is a professor at the University Medical Center Göttingen. Prof. Zweckstetter received three grants from the European Research Council (ERC) which helped his team to unravel the structure and function of proteins using the power of NMR spectroscopy.

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