Macromolecular assemblies, such as a microtubule shown to the right (Structure 2002, 10, 1317), perform many critical functions in a cell.
Structural information is required for understanding at the molecular and chemical levels how these machines work. However, their large
sizes make structure determination by NMR and X-ray crystallography difficult. Single particle cryo-electron microscopy (cryo-EM) is
becoming an important tool for studying such protein complexes. Uniquely it can reveal the overall protein structures without them having
to be crystallized, and it requires only a miniscule amount of sample. Cryo-EM can also be used for studying the conformational changes of
molecular assemblies. We use JEM-2010F TEM equipped with Gatan 4k x 4k CCD camera.
Eukaryotic DNA replication initiation
The origin recognition complex (ORC) is a six-protein ATPase machine conserved in all eukaryotes. The yeast ORC constitutively
binds to and marks the replication origin throughout the cell cycle. Licensing of the DNA replication origin starts when the cell
division cycle protein Cdc6 binds to ORC. In collaboration with Dr. Bruce Stillman in Cold Spring Harbor Lab and Dr. Christian
Speck in MRC Clinical Sciences Centre, we have revealed by cryo-EM that ORC has a bi-lobed half-ring architecture, and that Cdc6
completes the ring for subsequently loading of the replicative helicase (NSMB 2005, 12, 965; PNAS 2008, 105, 10326). We found that
the yeast ORC wraps around and bends the origin DNA (Structure 2012, 20, 534). Our current effort is to elucidate how ORC recruits
and then loads the MCM2-7 helicase onto the DNA.
Mycobacterium tuberculosis (Mtb) Pup-proteasome pathway
Proteasome is widespread in archaea and eukaryotes but very rare in bacteria. Recently, the Mtb proteasome is found to be required
for the bacteria to resist killing by the host immune system. We found that the Mtb proteasome and the associated ATPase are structurally
similar to their eukaryotic counterparts (Mol Microbiol 2005, 55, 561; Mol Microbiol 2006, 59, 1417, Structure 2009, 17, 1377), yet possess
unique assembly and gating mechanism (EMBOJ 2010, 29, 2037). We elucidated the structure basis for species-specific inhibition of the
Mtb proteasome inhibitor Oxathiazol-2-ones (Nature 2009, 461, 621). We further revealed that the protein degradation tag Pup, a
prokaryotic ubiquitin-like protein, is intrinsically disordered, but folds into an α-helix upon binding to and recognized by the
proteasomal ATPase (NSMB 2010, 17, 1352). We are continuing our collaboration with Drs. Carl Nathan and Gang Lin in Weill Cornell
Medical College and Dr. Heran Darwin in New York University.
Bacterial pilus biogenesis
The chaperone-usher pathway in Gram-negative bacteria is dedicated to the assembly of a large family of virulence-associated surface structures,
typically hair-like fibers termed pili or fimbriae. We found that the outer membrane usher forms oligomers in lipid bilayer (JMB 2004, 344, 1397),
but only one channel is used for secretion of the pilus fiber (Cell 2008, 133, 640). The recent structures of the FimD usher alone and in complex with
the FimCH chaperone-adhesin show surprisingly that the outer membrane β-barrel, long believed to be rigid, changes its shape from an oval in the
inactive form to a near circle in the activated form (Nature 2011, 474, 49). We collaborate with Dr. David Thanassi in Stony Brook University to
continue our study of the usher function in pilus assembly.
Human Gamma-secretase membrane complex
Gamma-secretase is an intramembranous aspartyl protease that is required for the processing of many membrane proteins, including Notch and amyloid
precursor protein. In collaboration with Drs. Dennis Selkoe and Michael Wolfe in Harvard Medical School, we determined the low-resolution structure of
γ-secretase by negative stain EM (PNAS 2006, 103, 6889). Our recent cryo-EM study reveals several low-density cavities in the presumed transmembrane
region of the complex structure that might be important for substrate entry and catalysis (JMB 2009, 385, 642). We are carrying out further structural
studies of the ubiquitous membrane complex.
Li H., and Stillman B. The origin recognition complex: a biochemical and structural view.
Subcell Biochem., 62:37-58 (2012).
Lu W., Chai Q., Zhong M., Yu L., Fang J., Wang T., Li H., Zhu H., and Wei Y. Assembling of AcrB Trimer in Cell Membrane.
J Mol Biol., 423(1):123-134 (Oct 12, 2012).
Sun J., Kawakami H., Zech J., Speck C., Stillman B., and Li H. Cdc6-induced conformational changes in ORC bound to origin DNA revealed by cryo-electron microscopy.
Structure, 20(3):534-544 (Mar 7, 2012).
Yang S., Wang T., Bohon J., Gagné M.È., Bolduc M., Leclerc D., and Li H. Crystal structure of the coat protein of the flexible filamentous papaya mosaic virus..
J Mol Biol., 422(2):263-73. Epub 2012 May 30. (Sep 14, 2012 )
Harada Y., Li H., Wall J.S., Li H., and Lennarz W.J. Structural studies and the assembly of the heptameric post-translational translocon complex.
J. Biol. Chem., 286(4):2956-2965 (Jan 28, 2011). [Epub 2010 Sep 8].
Phan G., Remaut H., Wang T., Allen W.J., Pirker K.F., Lebedev A., Henderson N.S., Geibel S., Volkan E., Yan J., Kunze M.B., Pinkner J.S., Ford B., Kay C.W., Li H., Hultgren S.J., Thanassi D.G., and Waksman G. Crystal structure of the FimD usher bound to its cognate FimC-FimH substrate.
Nature, 474(7349):49-53 (Jun 2 2011).
Burns K.E., Cerda-Maira F.A., Wang T., Li H., Bishai W.R., and Darwin K.H. "Depupylation" of prokaryotic ubiquitin-like protein from mycobacterial proteasome substrates.
Mol Cell., 239(5):821-827 (Sep 10, 2010).
Li D., Li H., Wang T., Pan H., Lin G., and Li H. Structural basis for the assembly and gate closure mechanisms of the Mycobacterium tuberculosis 20S proteasome.
EMBO J., 29(12):2037-2047 (Jun 16, 2010).
Li H., Zhang J., Vierstra R.D., and Li H. Quaternary organization of a phytochrome dimer as revealed by cryoelectron microscopy.
Proc Natl Acad Sci U S A., 107(24):10872-10877 (Jun 15, 2010).
Lin G., Li D., Chidawanyika T., Nathan C., and Li H. Fellutamide B is a potent inhibitor of the Mycobacterium tuberculosis proteasome.
Arch Biochem Biophys., 501(2):214-220 (Sep 15, 2010).
Qi S., Pang Y., Hu Q., Liu Q., Li H., Zhou Y., He T., Liang Q., Liu Y., Yuan X., Luo G., Li H., Wang J., Yan N., and Shi Y. Crystal structure of the Caenorhabditis elegans apoptosome reveals an octameric assembly of CED-4.
Cell, 141(3):446-457 (Apr 30, 2010).
Sun J., and Li H. How to operate a cryo-electron microscope.
Methods Enzymol., 481:231-249 (2010).
Wang T., Darwin K.H., and Li H. Binding-induced folding of prokaryotic ubiquitin-like protein on the Mycobacterium proteasomal ATPase targets substrates for degradation.
Nat. Struct. Mol. Biol., 17(11):1352-1357 (Nov 2010). [Epub 2010 Oct 17].
Evrin C., Clarke P., Zech J., Lurz R., Sun J., Uhle S., Li H., Stillman B., and Speck C. A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication.
Proc. Natl. Acad. Sci. U.S.A., 106(48):20240-20245 (2009).
Harada, Y., Li, Hua, Li H., and Lennarz, W. J. Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site.
Proceedings of the National Academy of Sciences USA, 106(17):6945-6949 (2009).
Hayashi, M. K., Tang, C., Verpelli, C., Narayanan, R., Stearns, M. H., Xu, R.-M., Li H., Sala, C., and Hayashi, Y. The postsynaptic density proteins Homer and Shank form a polymeric network structure.
Cell, 137(1):159-171 (2009).
Li H. and Thanassi, D. G. Use of a combined cryo-EM and X-ray crystallography approach to reveal molecular details of bacterial pilus assembly by the chaperone/usher pathway.
Current Opinion in Microbiology, 12(2):326-332 (2009).
Li H., Wolfe, M. S., and Selkoe, D. J. Toward structural elucidation of the γ-secretase complex.
Structure, 17(3):326-334 (March, 2009).
Lin G., Li D., de Carvalho L.P., Deng H., Tao H., Vogt G., Wu K., Schneider J., Chidawanyika T., Warren J.D., Li H.*, Nathan C.*.
(* co-corresponding authors) Inhibitors selective for mycobacterial versus human proteasomes.
Nature, 461(7264):621-626 (2009).
Osenkowski P., Li H., Ye W., Li D., Aeschbach L., Fraering P.C., Wolfe M.S., Selkoe D.J., and Li H. Cryoelectron microscopy structure of purified gamma-secretase at 12 Å resolution.
J. Mol. Biol., 385(2):642-652 Epub 2008 Nov 5. (2009).
Wang T., Li H., Lin G., Tang C., Li D., Nathan C., Darwin K.H., and Li H. Structural insights on the Mycobacterium tuberculosis proteasomal ATPase Mpa.
Structure, 17(10):1377-1385 (2009).
Cacquevel M., Aeschbach L., Osenkowski P., Li D., Ye W., Wolfe M.S., Li H., Selkoe D.J. and Fraering P.C. Rapid purification of active γ-secretase, an intramembrane protease implicated in Alzheimer’s disease.
J. Neurochem., 104(1):210-220 (2008).
Chen Z., Speck C., Wendel P., Tang C., Stillman B. and Li H. The architecture of the DNA replication origin recognition complex in Saccharomyces cerevisiae.
Proceedings of the National Academy of Sciences, 105(30):10326-10331 (2008).
Li Hua, Chavan M., Schindelin H., Lennarz W.J. and Li H. Structure of the oligosaccharide transferase complex at 12 Å resolution.
Structure, 16(3):432-440 (2008).
Remaut H., Tang C., Henderson N.S., Pinkner J.S., Wang T., Hultgren S.J., Thanassi D.G., Waksman G. and Li H. Fiber formation across the bacterial outer membrane by the chaperone/usher pathway.
Cell, 133(4):640-652 (2008).
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