We are a structural biology group. We use cryo-EM as our primary tool, in combination with other biophysical and biochemical means, to investigate the molecular mechanism of eukaryotic DNA replication, the mycobacterial Pup-proteasome system, and membrane proteins that are important to human health and diseases. Thanks to the recent advance in direct electron detector and image processing software, cryo-EM has become a high-resolution method for studying protein structure and dynamics.
Eukaryotic DNA replication
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 in G1 phase when the cell division cycle protein Cdc6 binds to ORC. Work in our lab has elucidated several key steps in origin activation and Mcm2-7 hexamer recruitment to DNA. We have revealed the architecture of ORC, 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). The inactive double hexamer is converted into two active helicase the Cdc45-Mcm2-7-GINs (CMG) complexes at the G1-S transition. In the S phase, CMG works together with polymerases to synthesize new DNA. Contrary to the widely held belief the polymerases trail behind the helicase, we found recently that the leading strand DNA polymerase epsilon rides ahead of the CMG helicase.
- 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.
- The origin recognition complex: a biochemical and structural view. Li H, Stillman B. Subcell Biochem. 2012, 62, 37-58. (Review).
- 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.
- 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.
- The architecture of a eukaryotic replisome. Sun J, Shi Y, Georgescu RE, Yuan Z, Chait BT, Li H, O’Donnell ME. Nature structural & molecular biology. 2015; 22, 976-82.
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. Using cryo-EM, X-ray crystallography, and protein biochemistry, 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 basis for species-specific inhibition of the Mtb proteasome inhibitor Oxathiazol-2-ones. We found 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. More recently, we found the ATP-independent proteasome activator PafE forms a dodecamer, unlike any previously known activators.
- 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.
- 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.
- 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.
- An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis. Jastrab JB, Wang T, Murphy JP, Bai L, Hu K, Merkx R, Huang J, Ovaa H, Gygi SP, Li H, Darwin KH. Proc Natl Acad Sci U S A. 2015; 112, E1763-72.
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 structural analysis of these complexes, as minimum amount of material is required and the method is compatible with many mild detergents. We have been studying the bacterial pilus assembly ushers, the yeast oligosaccharyl transferase complex that N-glycosylates the nascent polypeptide chains. Recently, we solved crystal structure of the ER-anchored Xxylt1 that O-glycosylates Notch EGF repeats, and resolved the reaction mechanism of a large family of retaining glycosyltransferases.
- 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.
- 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.
- 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.
- The pilus usher controls protein interactions via domain masking and is functional as an oligomer. Werneburg GT, Henderson NS, Portnoy EB, Sarowar S, Hultgren SJ, Li H, Thanassi DG. Nat Struct Mol Bio. 2015, 22, 540-6.
- Notch-modifying xylosyltransferase structures support an SNi-like retaining mechanism. Yu H, Takeuchi M, LeBarron J, Kantharia J, London E, Bakker H, Haltiwanger RS, Li H, Takeuchi H. Nat Chem Bio. 2015, 11, 847-54.