Research
We use cryoEM and structural mass spectrometry (especially H/D-exchange MS) to study the dynamic interplay of fusion proteins and membranes during virus entry. Cryo-electron tomography enables us to trigger fusion reactions then flash freeze them to capture and image transient intermediate protein-membrane interactions. We can thus image the 3-dimensional organization of protein-mediated fusion reactions. HDX-MS is exquisitely sensitive to protein dynamics and monitoring conformational changes as well as strain-specific differences between proteins that can be correlated with antigenic and functional phenotypes.
Mechanisms of Protein-mediated Membrane Fusion
Cryo-electron tomography and structural mass spectrometry are being used to understand how viral fusion proteins mediate the remodeling of host and viral membranes leading to formation of a fusion pore. Cryo-ET is perfectly suited for capturing transient intermediates and enabling them to be imaged in 3-dimensions. We can resolve membrane leaflets, intermediate protein conformations, and other viral components such as matrix layers that play key regulatory roles during fusion.
Benhaim MA, Mangala Prasad V, Garcia NK, Guttman M, Lee KK.
Science Advances 2020 Apr 29;6(18):eaaz8822. doi: 10.1126/sciadv.aaz8822.
Direct imaging of liquid domains in membranes by cryo-electron tomography.
Cornell CE, Mileant A, Thakkar N, Lee KK, Keller SL.
Proc Natl Acad Sci U S A. 2020 Aug 18;117(33):19713-19719. doi: 10.1073/pnas.2002245117.
Hom N, Gentles L, Bloom JD, Lee KK.
J Virol. 2019 Jun 14;93(13):e00161-19. doi: 10.1128/JVI.00161-19.
De novo design of tunable, pH-driven conformational changes.
Boyken SE, Benhaim MA, Busch F, Jia M, Bick MJ, Choi H, Klima JC, Chen Z, Walkey C, Mileant A, Sahasrabuddhe A, Wei KY, Hodge EA, Byron S, Quijano-Rubio A, Sankaran B, King NP, Lippincott-Schwartz J, Wysocki VH, Lee KK, Baker D.
Science. 2019 May 17;364(6441):658-664. doi: 10.1126/science.aav7897.
Influenza Virus-Liposome Fusion Studies Using Fluorescence Dequenching and Cryo-electron Tomography.
Gui L, Lee KK.
Methods Mol Biol. 2018;1836:261-279. doi: 10.1007/978-1-4939-8678-1_13.
Visualization and Sequencing of Membrane Remodeling Leading to Influenza Virus Fusion.
Gui L, Ebner JL, Mileant A, Williams JA, Lee KK.
J Virol. 2016 Jul 11;90(15):6948-6962. doi: 10.1128/JVI.00240-16.
Dynamic changes during acid-induced activation of influenza hemagglutinin.
Garcia NK, Guttman M, Ebner JL, Lee KK.
Structure. 2015 Apr 7;23(4):665-76. doi: 10.1016/j.str.2015.02.006.
Architecture of a nascent viral fusion pore.
Lee KK.
EMBO Journal. 2010 Apr 7;29(7):1299-311. doi: 10.1038/emboj.2010.13.
The structural and dynamic basis for antigenic variation in rapidly evolving viruses
Extreme variation is embodied in the surface antigens of viruses such as influenza, HIV, and hepatitis C virus. This variation is the primary impediment to development of broadly cross-reactive antibody responses. We are working to advance our understanding of structural and dynamic variation in HIV, influenza, and SARS-CoV-2 with the aim of using this information to identify the structural traits that underlie phenotypic differences in neutralization sensitivity and the ability to elicit a neutralizing antibody response.
While variation has long been appreciated at the sequence level, the resulting structural consequences are poorly understood. Recent high-resolution structures of trimers have provided a static blueprint of viral antigen architectures. Under native conditions however, the proteins are highly dynamic and exhibit structural fluctuations and large-scale, switching between states. If static structures reflect a protein's anatomy, structural dynamics, breathing, and conformational changes reflect its physiology. It is at this level that connections between structure and differences in neutralization sensitivity and transmissibility are expected to be most apparent.
Shipley MM*, Mangala Prasad V*, Doepker LE, Dingens AS, Ralph DK, Harkins E, Dhar A, Arenz D, Chohan V, Weight H, Mandaliya K, Bloom JD, Matsen Iv F, Lee KK†, Overbaugh JM†.
Elife. 2021 Jul 15;10:e68110. doi: 10.7554/eLife.68110.
Simonich CA, Doepker L, Ralph D, Williams JA, Dhar A, Yaffe Z, Gentles L, Small CT, Oliver B, Vigdorovich V, Mangala Prasad V, Nduati R, Sather DN, Lee KK, Matsen Iv FA, Overbaugh J.
Nature Commun. 2019 May 16;10(1):2190. doi: 10.1038/s41467-019-09481-7.
Epitope-Independent Purification of Native-Like Envelope Trimers from Diverse HIV-1 Isolates.
Verkerke HP, Williams JA, Guttman M, Simonich CA, Liang Y, Filipavicius M, Hu SL, Overbaugh J, Lee KK.
J Virol. 2016 Sep 29;90(20):9471-82. doi: 10.1128/JVI.01351-16.
HIV-1 Neutralizing Antibodies with Limited Hypermutation from an Infant.
Simonich CA, Williams KL, Verkerke HP, Williams JA, Nduati R, Lee KK, Overbaugh J.
Cell. 2016 Jun 30;166(1):77-87. doi: 10.1016/j.cell.2016.05.055.
Probing the Impact of Local Structural Dynamics of Conformational Epitopes on Antibody Recognition.
Liang Y, Guttman M, Davenport TM, Hu SL, Lee KK.
Biochemistry. 2016 Apr 19;55(15):2197-213. doi: 10.1021/acs.biochem.5b01354.
Antibody potency relates to the ability to recognize the closed, pre-fusion form of HIV Env.
Guttman M, Cupo A, Julien JP, Sanders RW, Wilson IA, Moore JP, Lee KK.
Nature Commun. 2015 Feb 5;6:6144. doi: 10.1038/ncomms7144.
Host-pathogen interactions and immune responses
Despite the high levels of variation in rapidly evolving viruses, remarkably, the host immune system in some rare cases is able to mount a broadly neutralizing response that blocks infection by highly divergent strains of HIV or influenza for example. Through collaborations with groups at the Fred Hutchinson Cancer Research Center, University of Washington, Vaccine Research Center NIH, and other institutions, we are analyzing these types of special, broadly neutralizing antibodies as well as vaccine candidates that seek to elicit similarly broad, potent immune responses.
Shipley MM*, Mangala Prasad V*, Doepker LE, Dingens AS, Ralph DK, Harkins E, Dhar A, Arenz D, Chohan V, Weight H, Mandaliya K, Bloom JD, Matsen Iv F, Lee KK†, Overbaugh JM†.
Elife. 2021 Jul 15;10:e68110. doi: 10.7554/eLife.68110.
Quadrivalent influenza nanoparticle vaccines induce broad protection.
Boyoglu-Barnum S, Ellis D, Gillespie RA, Hutchinson GB, Park Y-J, Moi SM, Acton OJ, Ravichandran R, Murphy M, Pettie D, Matheson N, Carter L, Creanga A, Watson MJ, Kephart S, Ataca S, Vaile JR, Ueda G, Crank M, Stewart L, Lee KK, Guttman M, Baker D, Mascola JR, Veesler D, Graham BS, King NP, Kanekiyo M.
Nature 2021 Mar 24; 10.1038/s41586-021-03365-x.
Williams JA, Gui L, Hom N, Mileant A, Lee KK.
J Virol. 2018 Feb 26;92(6):e02006-17. doi: 10.1128/JVI.02006-17.
Davenport TM, Gorman J, Joyce MG, Zhou T, Soto C, Guttman M, Moquin S, Yang Y, Zhang B, Doria-Rose NA, Hu SL, Mascola JR, Kwong PD, Lee KK.
Structure. 2016 Aug 2;24(8):1346-1357. doi: 10.1016/j.str.2016.06.012.
HIV-1 Neutralizing Antibodies with Limited Hypermutation from an Infant.
Simonich CA, Williams KL, Verkerke HP, Williams JA, Nduati R, Lee KK, Overbaugh J.
Cell. 2016 Jun 30;166(1):77-87. doi: 10.1016/j.cell.2016.05.055.
Antibody potency relates to the ability to recognize the closed, pre-fusion form of HIV Env.
Guttman M, Cupo A, Julien JP, Sanders RW, Wilson IA, Moore JP, Lee KK.
Nature Commun. 2015 Feb 5;6:6144. doi: 10.1038/ncomms7144.
