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Huilin Li

Research Interests

  • Macromolecular assemblies, such as a microtubule shown to the right (Structure 2002), 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. Thanks to the advent of direct electron detector and novel computational image reconstruction algorithm, single particle cryo-electron microscopy (cryo-EM) has become a high-resolution tool for studying structure and conformational dynamics of protein complexes. Our lab has been using cryo-EM combined with other biophysical and biochemical methods to study the eukaryotic DNA replication system and the Mycobacterium tuberculosis Pup-proteasome system over the past decade.
  • Eukaryotic DNA replication:
    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 in G1 phase when the cell division cycle protein Cdc6 binds to ORC. In collaboration with Dr. Bruce Stillman and Dr. Christian Speck, 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; PNAS 2008). ORC-Cdc6 is the active loading platform that recruits the cdt1-bound Mcm2-7 hexamer one at a time (NSMB 2013), and that one ORC-Cdc6, not two, recruits two Mcm2-7 hexamers to form the inactive double-hexamer encircling dsDNA (G&D 2014). At G1/S transition, the double-hexamer is converted to two active helicases the Cdc45-Mcm2-7-GINS (CMG) complexes.

    In S phase, the active CMG helicase (CMG) works with the leading strand polymerase epsilon, the lagging strand polymerase delta, and the primase-polymerase alpha to synthesize new DNA. In collaboration with Dr. Michaeal O’Donnell, we have found the leading strand polymerase epsilon rides ahead of the helicase, rather than trailing behind the helicase as widely believed (NSMB 2015).

    1. 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.
    2. The origin recognition complex: a biochemical and structural view. Li H, Stillman B. Subcell Biochem. 2012, 62, 37-58. (Review).
    3. 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.
    4. 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.
    5. 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 (Mtb) 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 (Structure 2009, EMBO J 2010). We elucidated the structure basis for species-specific inhibition of the Mtb proteasome inhibitor Oxathiazol-2-ones (Nature 2009). 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). Recently, Heran Darwin lab discovered an ATP-independent Mtb proteasomal activator PafE, which surprisingly formed a dodecameric ring, as demonstrated by EM (PNAS 2015). Our work may help structure-based anti-TB chemotherapeutic development targeting the Pup-proteasome system.

    1. 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.
    2. 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.
    3. 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.
    4. 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.
    5. 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.
  • Membrane-embedded enzyme complexes
    Membrane proteins, in particular eukaryotic membrane proteins, are underrepresented in the protein structural database. This is so because it is often difficult to produce sufficient material for traditional protein crystallography, and membrane complex is generally sensitive to the detergents used for solubilization and purification. Cryo-EM is uniquely suited for structural analysis of membrane complexes, as minimum amount of material is required and the method is compatible with many mild detergents. We have analyzed the structures of the bacterial pilus assembly ushers (Cell 2008, Nature 2011, NSMB 2015), the yeast oligosaccharyl transferase complex that N-glycosylates the nascent polypeptide chains (Structure 2008), and the ER-anchored Xxylt1 that O-glycosylates the Notch receptor (Nat Chem Bio 2015).

    1. 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.
    2. 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.
    3. 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.
    4. 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.
    5. 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.

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