Thanks to the recent advance in electron detection hardware and image processing software, cryo-EM is revolutionizing structural biology, particularly in the area of large protein machines and multi-subunit membrane complexes. We have been using cryo-EM as our primary tool, in combination with other biophysical and biochemical means, to investigate the molecular mechanism of DNA replication, the bacterial Pup-proteasome system, and several membrane complexes that are essential to human health and diseases.
- Structural biology: Ion channel seen by electron microscopy. Henderson R. Nature. 2013, 504, 93-4.
- The resolution revolution. Kühlbrandt W. Science. 2014, 343, 1443-4.
Eukaryotic DNA replication initiation
Eukaryotic chromosomal replication initiation is an intricate process that requires the coordinated and tightly regulated action of numerous molecular machines. Failure to ensure once only replication initiation per cell cycle can result in uncontrolled proliferation and genomic instability, two hallmarks of tumor genesis. The origin recognition complex (ORC), first discovered in yeast in Dr. Bruce Stillman lab at Cold Spring Harbor Laboratory in 1992, is a six-protein ATPase machine conserved in all eukaryotes. 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 Cdc6p binds to ORC. Work in our lab has elucidated several key steps in origin activation and Mcm2-7 hexamer recruitment on to DNA. We have revealed the architecture of ORC, how ORC wraps around the origin DNA, how Cdc6 completes the ORC ring and activates it for subsequently loading of the replicative helicase, how ORC binds to and cracks open and then loads the Mcm2-7 helicase core onto DNA, how two Mcm2-7 hexamers assemble on DNA to form the Mcm2-7 double-hexamer, the pre-replication complex (pre-RC). Our research has advanced the field of eukaryotic DNA replication.
- Structural and mechanistic insights into Mcm2-7 double-hexamer assembly and function. Sun J, Fernandez-Cid A, Riera A, Tognetti S, Yuan Z, Stillman B, Speck C, Li H. Genes Dev. 2014, 28, 2291-303.
- Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA. Sun J, Evrin C, Samel SA, Fernández-Cid A, Riera A, Kawakami H, Stillman B, Speck C, Li H. Nat Struct Mol Biol. 2013, 20, 944-51.
- The origin recognition complex: a biochemical and structural view. Li H, Stillman B. Subcell Biochem. 2012, 62, 37-58. (Review).
- Cdc6-induced conformational changes in ORC bound to origin DNA revealed by cryo-electron microscopy. Sun J, Kawakami H, Zech J, Speck C, Stillman B, Li H. Structure. 2012, 20, 534-44.
- The architecture of the DNA replication origin recognition complex in Saccharomyces cerevisiae. Chen Z, Speck C, Wendel P, Tang C, Stillman B, Li H. Proc Natl Acad Sci U S A. 2008, 105, 10326-31.
Mycobacterium tuberculosis Pup-proteasome system
Tuberculosis kills 1.5-2 million people globally every year. An effective vaccine or chemotherapy has yet to be developed. Recently, through a large-scale transposon mutagenesis screening, the Mycobacterium tuberculosis (Mtb) proteasome and Mtb proteasomal ATPase (Mpa) were found to be required for Mtb resistance to killing by a source of nitric oxide (NO). NO is required by the host immune system to control Mtb infections. Proteasome and Mpa appear to protect Mtb against NO by degrading proteins after exposure to NO. Thus, Mpa and the Mtb proteasome may be promising targets for the development of anti-Tb chemotherapeutics. We have combined cryo-EM, X-ray crystallography, and protein biochemistry to elucidate the structure and function of the Mtb proteasome, Mpa ATPase, respectively. We found that the Mtb proteasome and the associated ATPase are structurally similar to their eukaryotic counterparts yet possess unique assembly and gating mechanism. We elucidated the structure basis for species-specific inhibition of the Mtb proteasome inhibitor Oxathiazol-2-ones. 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. Our work is setting the stage for the structure-based anti-TB chemotherapeutic development targeting the Pup-proteasome system.
- Binding-induced folding of prokaryotic ubiquitin-like protein on the Mycobacterium proteasomal ATPase targets substrates for degradation. Wang T, Darwin KH, Li H. Nat Struct Mol Biol. 2010, 17, 1352-7.
- Structural basis for the assembly and gate closure mechanisms of the Mycobacterium tuberculosis 20S proteasome. Li D, Li H, Wang T, Pan H, Lin G, Li H. EMBO J. 2010, 29, 2037-47.
- Structural insights on the Mycobacterium tuberculosis proteasomal ATPase Mpa. Wang T, Li H, Lin G, Tang C, Li D, Nathan C, Darwin KH, Li H. Structure. 2009, 17, 1377-85.
- Inhibitors selective for mycobacterial versus human proteasomes. Lin G, Li D, de Carvalho LP, Deng H, Tao H, Vogt G, Wu K, Schneider J, Chidawanyika T, Warren JD, Li H, Nathan C. Nature. 2009, 461, 621-6.
- Structure of the Mycobacterium tuberculosis proteasome and mechanism of inhibition by a peptidyl boronate. Hu G, Lin G, Wang M, Dick L, Xu RM, Nathan C, Li H. Mol Microbiol. 2006, 59, 1417-28.
Cryo-EM structural biology of membrane protein complexes
Membrane proteins, in particular the eukaryotic membrane proteins, are underrepresented in the protein structural database. This is so because it is very difficult to produce sufficient material for traditional protein crystallography, and the structure of a membrane protein complex is generally sensitive to the detergents used for solubilization and purification. Cryo-EM is uniquely suited for determining the medium- to high-resolution structures of these complexes, as minimum amount of material is required and the method is compatible with many mild detergents. We have been studying by cryo-EM the bacterial pilus assembly ushers, the yeast oligosaccharyl transferase complex that glycosylates the nascent polypeptide chains, and the human γ-secretase that processes many membrane proteins including the Notch receptor and amyloid precursor protein.
- Crystal structure of the FimD usher bound to its cognate FimC-FimH substrate. Phan G, Remaut H, Wang T, Allen WJ, Pirker KF, Lebedev A, Henderson NS, Geibel S, Volkan E, Yan J, Kunze MB, Pinkner JS, Ford B, Kay CW, Li H, Hultgren SJ, Thanassi DG, Waksman G. Nature. 2011, 474, 49-53.
- Cryoelectron microscopy structure of purified gamma-secretase at 12 A resolution. Osenkowski P, Li H, Ye W, Li D, Aeschbach L, Fraering PC, Wolfe MS, Selkoe DJ, Li H. J Mol Biol. 2009, 385, 642-52.
- Structure of the oligosaccharyl transferase complex at 12 A resolution. Li H, Chavan M, Schindelin H, Lennarz WJ, Li H. Structure. 2008, 16, 432-40.
- Fiber formation across the bacterial outer membrane by the chaperone/usher pathway. Remaut H, Tang C, Henderson NS, Pinkner JS, Wang T, Hultgren SJ, Thanassi DG, Waksman G, Li H. Cell. 2008, 133, 640-52.
- The outer membrane usher forms a twin-pore secretion complex. Li H, Qian L, Chen Z, Thibault D, Liu G, Liu T, Thanassi DG. J Mol Biol. 2004, 344, 1397-407.
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