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High-Resolution Structural Biology of Membrane Protein Complexes in Their Native Environment

• Doreen Matthies, PhD, Head, Unit on Structural Biology
• Fei Zhou, PhD, Scientist
• So-Young Kim, Visiting Postdoctoral Fellow
• Louis Tung Faat Lai, PhD, Visiting Postdoctoral Fellow
• Elissa J. Moller, BS, Graduate Student

We are interested in the structure and function of membrane protein complexes in their native lipid-membrane environment to understand their mechanism and the influence of their immediate surrounding and how these affect human health and disease. A cell contains many different lipid membranes with various lipid contents and distributions, which are very important for a membrane’s morphology and function. However, very little is understood about how the various micro-environments are formed and maintained and how they influence the structure and function of membrane proteins. Studying membrane protein complexes in their native biological membrane is therefore required.

We use a combination of molecular biology, biochemistry, and biophysical methods to study molecular transport across membranes, with a focus on how the immediate native environment influences the structure and function of membrane proteins, but also how proteins and lipids shape and functionalize a lipid membrane.

Cryo-electron microscopy (Cryo-EM) is one of the main structural-biology methods of the lab. Using single-particle Cryo-EM, we solve high-resolution structures of membrane proteins in artificial environments such as in detergent micelles and lipid nano-discs. However, we are extending this approach to studying membrane proteins in their native environment, using native lipid nano-discs, membrane fractions in forms of vesicles, and intact cells and tissues, using a combination of correlative light and electron-microscopy techniques, including cryo-fluorescent microscopy, cryo-focused ion beam-scanning electron microscopy (Cryo-FIBSEM), Cryo-EM, and cryo-electron tomography (Cryo-ET).

Structure and function of magnesium channels

Magnesium (Mg²⁺) is the most abundant divalent cation inside cells, with an average Mg²⁺ concentration of about 20 mM, most of it bound to proteins and ATP. Magnesium plays an essential role in cellular physiology, acting as a cofactor for more than 600 enzymes, including protein kinases, ATPase, exonucleases, and other nucleotide-related enzymes. Deficiency in Mg²⁺ is associated with diseases such as muscular dysfunction, bone wasting, immunodeficiency, cardiac syndromes, and neuronal disorders. The bacterial magnesium channel CorA is a homo-pentameric channel, which forms a symmetric closed state at normal to high concentrations of magnesium, with magnesium-binding sites between protomers as well as near the membrane pore. At low magnesium concentrations, the channel undergoes an asymmetric opening, which is likely to be caused by the destabilization of protomer interactions when magnesium ions dissociate from their binding site. Louis Lai is expanding the research on magnesium channels, including looking at eukaryotic magnesium channels. To investigate the structure and mechanism of these channels, structural studies on synthetic and native nano-discs, as well as in liposomes are planned.

Structural determination of the full-length SARS-CoV-2 spike protein and drug development

COVID-19, caused by the SARS-CoV-2 virus, has posed a global threat since it was first identified at the end of 2019. The rapid development of vaccines has helped counteract the severeness of the disease. However, vaccines for children under the age of 12 have only just been approved. More children have been infected by more contagious variants of the virus, and the recent surge in COVID-19 cases has put an unprecedented pressure on the pediatric health care system. The SARS-CoV-2 spike protein is responsible for the initial binding of the virus to the ACE2 receptor on human cells. Better understanding of the function and structure of the spike protein is critical for primary prevention and for the development of a vaccine and therapeutic treatments to combat the COVID-19 pandemic. Structures of the spike protein’s soluble ectodomain have been determined, but the full-length spike, including its membrane domain, have not been well studied. We are working towards determining the structures of full-length spike protein complexes and identifying the key vaccine- and drug-binding interfaces in order to develop treatments that block viral entry into human cells with high efficiency and specificity, and which are also safe for children. Fei Zhou has successfully cloned and expressed the full-length spike protein, and we are working towards high-resolution structural determination of different variants and complexes.

Collaborations

Our collaborations involve structural and computational studies on a variety of membrane-protein complexes, including transporters, channels, and receptors, virus-like particles (VLP), SARS-CoV-2 accessory membrane proteins, extracellular vesicles, and lipid transport across cells, as well as novel detergents and polymers to gently extract membrane-protein complexes from their native lipid environment for high-resolution structural studies.

Publications

1. He S, Chou H-T, Matthies D, Wunder T, Meyer MT, Atkinson N, Martinez-Sanchez A, Jeffrey PD, Port SA, Patena W, He G, Chen VK, Hughson FM, McCormick AJ, Mueller-Cajar O, Engel BD, Yu Z, Jonikas MC. The structural basis of Rubisco phase separation in the pyrenoid. Nat Plants 2020 6(12):1480–1490.
2. Qiu B, Matthies D, Fortea E, Yu Z, Boudker O. Cryo-EM structures of excitatory amino acid transporter 3 visualize coupled substrate, sodium, and proton binding and transport. Sci Adv 2021 7:1–9.
3. Wasmuth EV, Broeck AV, LaClair JR, Hoover EA, Lawrence KE, Paknejad N, Pappas K, Matthies D, Wang B, Feng W, Watson PA, Zinder JC, Karthaus WR, de la Cruz MJ, Hite RK, Manova-Todorova K, Yu Z, Weintraub ST, Klinge S, Sawyers CL. Allosteric interactions prime androgen receptor dimerization and activation. Mol Cell 2022 82:2021–2031.
4. Petersen J, Lu J, Fitzgerald W, Zhou F, Blank PS, Matthies D, Zimmerberg J. Unique aggregation of retroviral particles pseudotyped with the delta variant SARS-CoV-2 spike protein. Viruses 2022 14:1024.

Collaborators

• Tamir Gonen, PhD, Laboratory of Molecular Electron Microscopy, UCLA, Los Angeles, CA
• Rick K. Huang, PhD, NIH Intramural Research CryoEM Consortium, NCI, Bethesda, MD
• Maria S. Ioannou, PhD, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada
• Marzena E. Pazgier, PhD, Uniformed Services University of the Health Sciences, Bethesda, MD
• Alexander J. Sodt, PhD, Unit on Membrane Chemical Physics, NICHD, Bethesda, MD
• Gisela Storz, PhD, Section on Environmental Gene Regulation, NICHD, Bethesda, MD
• Sergei I. Sukharev, PhD, University of Maryland, College Park, MD
• Joshua J. Zimmerberg, MD, PhD, Section on Integrative Biophysics, NICHD, Bethesda, MD
• Manuela Zoonens, PhD, Institut de Biologie Physico-Chimique, Centre National de la Recherche Scientifique, Paris, France

Contact

For more information, email doreen.matthies@nih.gov or visit https://www.nichd.nih.gov/research/atNICHD/Investigators/matthies.

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