On‐Cell NMR Contributions to Membrane Receptor Binding
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Minireviews ChemPlusChem doi.org/10.1002/cplu.202100134 On-Cell NMR Contributions to Membrane Receptor Binding Characterization Theresa Höfurthner, Borja Mateos, and Robert Konrat*[a] ChemPlusChem 2021, 86, 938–945 938 © 2021 The Authors. ChemPlusChem published by Wiley-VCH GmbH Wiley VCH Mittwoch, 23.06.2021 2106 / 207714 [S. 938/945] 1 Minireviews ChemPlusChem doi.org/10.1002/cplu.202100134 NMR spectroscopy has matured into a powerful tool to by membrane receptors on the cell surface; therefore, charac- characterize interactions between biological molecules at terizing their interactions at atomic resolution by NMR, here- atomic resolution, most importantly even under near to native after referred as on-cell NMR, can provide valuable mechanistic (physiological) conditions. The field of in-cell NMR aims to study information. This review aims to summarize recent on-cell NMR proteins and nucleic acids inside living cells. However, cells tools that give information about the binding site and the interrogate their environment and are continuously modulated affinity of membrane receptors to their ligands together with by external stimuli. Cell signaling processes are often initialized potential applications to in vivo drug screening systems. 1. Introduction and their interactions in the near to native environment. It should not be confused with in vivo NMR, which refers to One of the most important features of living organisms is the interactions studies with living organisms, e.g. MRI. Addition- bidirectional communication capability to respond to their ally, with ex vivo NMR, important information about how the environment by reacting to stimuli from the extracellular space drug is affected under physiological conditions can be achieved, and respond by producing and secreting signaling molecules to as a drug might not be able to cross the cellular membrane, shape their surroundings. The essential response system might be pumped out of the cell, is metabolized or interacts comprises different membrane proteins embedded in the with other cellular components instead of the therapeutic plasma membranes of cells. The extracellular parts of mem- target.[6] Ex vivo NMR divides into investigations inside (in-cell brane receptors typically interact with a variety of molecules, NMR), at the membrane (on-cell NMR) and outside (ex-cell NMR) such as proteins, neurotransmitters, hormones, metabolites, of the living cell. In this review, we focus on the studies of ligands, and ions. Upon binding, the membrane receptor membrane receptors and their interactions by on-cell NMR. We activates different physiological processes by triggering signal- will address important considerations covering the expression ing cascades. Given their eminent biological functionalities, of membrane receptors with living mammalian cells, quantifica- they are attractive targets for many drug discovery projects. For tion of ligand binding, binding site mapping, and ultimately example, approximately 30% of all protein-coding genes in determination of binding poses. The aim of this mini-review is humans account for integral membrane proteins and 40% of all to guide the reader through the opportunities and difficulties of FDA-approved drugs target membrane proteins[1].[2] on-cell NMR and eventually simplify their entry to the new field. The structural and biochemical characterization of mem- brane proteins and their binding partners is still challenging, although various improvements in fields of sample preparation, 2. NMR in Biological Environments hardware and data processing have been accomplished in recent years.[3] Specifically, X-ray crystallography and cryo-EM 2.1. The cellular environment have been established as the methods of choice for large macromolecules, as they provide structural information about Typical structural biology projects are carried out in solutions macromolecules with high-resolution. containing few salts in a buffer that adjusts the pH. The cellular A unique method to probe molecular interactions and environment, however, is way more complex and contains a dynamics at atomic resolution, however, is nuclear magnetic plethora of other molecules such as metabolites, osmolytes, resonance (NMR), be it in the solid-state (SS-NMR) or liquid-state other proteins, nucleic acids, lipids and polymeric assemblies, (LS-NMR). Therefore, it has emerged as an indispensable tool in depending on the specifics of the cellular (sub)-system.[7] The drug discovery, as NMR can also detect changes of the cell’s interior, for example, is highly crowded, which influences conformation and dynamics upon ligand binding, providing not only the translational and rotational diffusion of the unique opportunities to target allosteric binding sites.[4,5] Most molecules in the cell but also the function of biomolecules.[9,10] importantly, the results of in vitro assays might be misleading or Several NMR studies provided insightful information on the inconclusive as the functionally relevant molecular interactions protein stability and function in crowded cellular occur either in the cell’s cytoplasm, at the external surface of conditions.[9,11–13] Additionally, subcellular compartments, like living cells or in the extracellular matrix (ECM). Ex vivo NMR cytoplasm, nucleus, mitochondria and lysosomes, confine the involves interaction studies with cells, tissues or organs and cell’s volume. Crowding and confinement have important therefore, provides unique possibilities to study biomolecules consequences for protein stability and reactivity, as proteins can interact with cellular milieu molecules, either by excluded [14] [a] T. Höfurthner, Dr. B. Mateos, Prof. R. Konrat volume of preferential interactions, and are altered by post- Department of Structural and Computational Biology translational modifications (PTMs).[15] For more details on in-cell Max Perutz Laboratories, University of Vienna Vienna Biocenter Campus 5, 1030 Vienna (Austria) NMR, we recommend other reviews that focus on this [7,16–18] E-mail: [email protected] topic. © 2021 The Authors. ChemPlusChem published by Wiley-VCH GmbH. This is When investigating membrane receptor interactions on the an open access article under the terms of the Creative Commons Attribution cell surface the details of the extracellular matrix has also to be Non-Commercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used considered, as it contains a significant number of polysacchar- for commercial purposes. ides (both in free form or as a part of protein glycosylation ChemPlusChem 2021, 86, 938–945 www.chempluschem.org 939 © 2021 The Authors. ChemPlusChem published by Wiley-VCH GmbH Wiley VCH Mittwoch, 23.06.2021 2106 / 207714 [S. 939/945] 1 Minireviews ChemPlusChem doi.org/10.1002/cplu.202100134 sites), important for several molecular recognition events, and method of choice, because it is a versatile platform to monitor secondary metabolites that are relevant for intercellular both the presence of reporter molecules of cellular fate and the communication.[19] Therefore, the ECM not only supports the expression of specific receptor types using monoclonal anti- mechanical stability of the cells but also is essential for outside- bodies. For example, it is possible to detect the presence of pre- in or inside-out signalling mechanism.[20] apoptotic cells with the annexin V assay as Annexin V binds to phosphatidylserines that migrates to the outer plasma mem- brane in apoptosis. In parallel, cell viability can be checked by 2.2. Receptor characterization propidium iodide or trypan blue staining. Thus, in a single assay, several reporters of viability, membrane receptor status When investigating membrane receptors in mammalian cells, it and cellular phenotype can be monitored. is important to know precisely the identity and quantity of the An essential prerequisite for the recording of NMR experi- protein. Both can be achieved by using mass spectrometry, ments with living cells is to maintain cell viability. To this end, commercial available microarrays and RNA sequencing.[21] Addi- the usage of bioreactors that enable the continuous exchange tionally, as a negative control, a cell line not expressing the of media in the NMR instruments have been proposed,[26–30] or membrane receptor should be established. To this end, CRISPR/ alternatively cells can be encapsulated within agar or alginate Cas9 technology has emerged as an excellent tool to manipu- hydrogels.[27] The problem, however, associated with these late the genome of cells.[22] With that methodology, tailored cell approaches is the fact that highly viscous gels are not suitable lines can be designed to address a specific question involving for LS-NMR as they prevent the free tumbling of the protein the membrane receptor of interest. This method has a great under study in solution. To circumvent this problem our potential in mimicking disease-related conditions by introduc- laboratory has recently developed an alternative experimental ing point mutations, gene deletion or insertions.[23] Furthermore, strategy involving methylcellulose hydrogels to prevent cells CRISPR/Cas can be used in designing and developing new from sedimentation. This approach ensures proper tumbling of drugs, especially for GPRCs, as they have a highly selective extracellular components, while retaining the cells attached to a pattern of expression and activity levels in healthy and disease (hydrogel) scaffold. Depending