The Li Lab currently pursues three lines of research: eukaryotic DNA replication (project 1), the mycobacterial Pup-proteasome system (project 2), and protein glycosylation (project 3), as described below.
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 tumorigenesis. The origin recognition complex (ORC) is a six-protein 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 critical cell division cycle protein Cdc6p binds to ORC in the G1 phase of the cell cycle.
In collaboration with the labs of Dr. Christian Speck and Dr. Bruce Stillman, we have mapped the architecture of ORC (Chen et al 2008 Proc Natl Acad Sci U S A), captured several key intermediates during origin activation and Mcm2-7 hexamer recruitment on to DNA (Sun et al 2013 Nat Struct Mol Biol, Sun et al 2014 Genes Dev), and elucidated how Cdc6 completes the ORC ring and activates it to load the replicative helicase (Yuan et al 2017 Nat Struct Mol Biol). Most recently, we showed how the two Mcm2-7 hexamers come together on the origin DNA to form the pre-replication complex—the Mcm2-7 double-hexamer (Noguchi et al 2017 Proc Natl Acad Sci U S A).
In the S phase of the cell cycle, the active CMG helicase 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. Michael O’Donnell’s lab, we have determined the structure of the 11-protein CMG helicase (Yuan et al 2016 Nat Struct Mol Biol) and its complex with a forked DNA (Georgescu et al 2017 Proc Natl Acad Sci U S A), and mapped a few key components of the replisome (Sun et al 2015 Nat Struct Mol Biol). We found Ctf4 trimer is able to coordinate two CMG helicases and one Pol alpha-primase into a replication factory core (Yuan et al 2019 eLife). Our work has advanced the field of eukaryotic DNA replication (Li et al 2018 BioEssay; O’Donnell et al NSMB 2018).
Chen Z, Speck C, Wendel P, Tang C, Stillman B, Li H. 2008. The architecture of the DNA replication origin recognition complex in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 105(30):10326–10331.
Sun J, Evrin C, Samel S, Fernadez-Cid A, Riera A, Kawakami H, Zech J, Stillman B, Speck C, Li H. 2013. Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA. Nat Struct Mol Biol 20(8):944–951.
Sun J, Fernandez-Cid A, Riera A, Tognetti S, Yuan Z, Stillman B, Speck C, Li H. 2014. Structural and mechanistic insights into licensing of DNA replication. Genes Dev 28:2291–2303.
Sun J, Shi Y, Georgescu RE, Yuan Z, Chait BT, Li H*, O’Donnell ME*. 2015. The architecture of a eukaryotic replisome. Nat Struct Mol Biol 22(12):976–982.
Yuan Z, Bai L, Sun J, Georgescu R, O’Donnell ME, Li H. 2016. Structure of the eukaryotic replicative CMG helicase suggests a pumpjack motion for translocation. Nat Struct Mol Biol 23(3):217–224.
Noguchi Y, Yuan Z, Bai L, Schneider S, Zhao G, Stillman B, Speck C, Li H. 2017. Cryo-EM structure of Mcm2-7 double hexamer on DNA suggests a lagging-strand DNA extrusion model. Proc Natl Acad Sci U S A 114(45):E9529–E9538.
Yuan Z, Riera A, Bai L, Sun J, Nandi S, Spanos C, Chen ZA, Barbon M, Rappsilber J, Stillman B, Speck C, Li H. 2017. Structural basis of MCM2-7 replicative helicase loading by ORC-Cdc6 and Cdt1. Nat Struct Mol Biol 24(3):316–324.
Georgescu R, Yuan Z, Bai L, de Luna Almeida Santos R, Sun J, Zhang D, Yurieva O, Li H*, O’Donnell ME*. 2017. Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation. Proc Natl Acad Sci U S A 114(5):E697–E706.
Li H, O’Donnell ME. 2018. The eukaryotic CMG helicase at the replication fork: Emerging architecture reveals an unexpected mechanism. BioEssays 40(3).
O’Donnell ME, Li H. 2018. The ring-shaped hexameric helicases that function at DNA replication forks. Nat Struct Mol Biol 25(2):122–130.
Yuan Z, Georgescu R, Santos RLA, Zhang D, Bai L, Yao NY, Zhao G, O’Donnell ME, Li H. 2019. Ctf4 organizes sister replisomes and Pol α into a replication factory. eLife e47405.
Tuberculosis (TB) kills 1.5–2 million people globally every year. In the U.S. alone, 13 million people have latent TB infection; and about 10 percent of them will develop active TB disease during their lifetime. An effective vaccine or chemotherapy has yet to be developed. Mycobacterium tuberculosis (Mtb) — the bacterium that causes TB — has evolved a Pup-proteasome system that is functionally analogous to but chemically distinct from the eukaryotic Ubiquitin-proteasome system. The Pup-proteasome is 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. Thus, the Pup-proteasome system is a promising target for the development of anti-Tb chemotherapeutics.
In collaboration with the labs of Dr. Carl Nathan, Dr. Gang Lin and Dr. Heran Darwin, we have combined cryo-EM, X-ray crystallography and protein biochemistry to elucidate the structure and function of the bacterial system. We found that the Mtb proteasome is structurally similar to the eukaryotic proteasome yet possesses unique assembly and gating mechanism (Hu et al 2006 Mol Microbiol; Li et al 2010 EMBO J). Like its eukaryotic counterpart, the bacterial proteasome can be activated either by an ATP-dependent activator (Mpa) (Wu et al 2017 Mol Microbiol), or by an ATP-independent activator (PafE) (Jastrab et al 2015 Proc Natl Acad Sci U S A; Bai et al 2016 Proc Natl Acad Sci U S A; Hu et al 2018 J Biol Chem). Interestingly, 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 Mpa (Wang et al 2010 Nat Struct Mol Biol). To facilitate the development of anti-TB chemotherapeutics, we have elucidated the structural basis for species-specific inhibition of the Mtb proteasome by oxathiazol-2-ones (Lin et al 2009 Nature), by several N,C-capped dipeptides (Hsu et al 2017 Biochemistry), and by the phenylimidazo-based inhibitors (Zhan et al 2019 J Med Chem). Remarkably, some of the dipeptide variants exhibited potent and selective inhibition to the human immunoproteasome (Santos et al 2017 Nat Commun).
Hu G, Lin G, Wang M, Dick L, Xu RM, Nathan C, Li H. 2006. Structure of the Mycobacterium tuberculosis proteasome and mechanism of inhibition by a peptidyl boronate. Mol Microbiol 59(5):1417–1428.
Lin G*, Li D, de Carvalho L, Deng H, Tao H, Vogt G, Wu K, Schneider J, Chidawanyika T, Warren JD, Li H*, Nathan C*. 2009. Inhibitors selective for mycobacterial versus human proteasomes. Nature 461(7264):621–626.
Wang T, Darwin KH, Li H. 2010. Binding-induced folding of prokaryotic ubiquitin-like protein (Pup) targets substrates for proteasomal degradation. Nat Struct Mol Biol 17(11):1352–1357.
Li D, Li H, Wang T, Pan H, Lin G, Li H. 2010. Structural basis for the assembly and gate closure mechanisms of the Mycobacterium tuberculosis 20S proteasome. EMBO J. 29(12):2037–2047.
Jastrab JB, Wang T, Murphy JP, Bai L, Hu K, Merkx R, Huang J, Chatterjee C, Ovaa H, Gygi SP, Li H, Darwin KH. 2015. An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis. Proc Natl Acad Sci U S A. 112(14):E1763–1772.
Bai L, Hu K, Wang T, Jastrab JB, Darwin KH, Li H. 2016. Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 113(14):E1983–1992.
Wu Y, Hu K, Li D, Bai L, Yang S, Jastrab JB, Xiao S, Hu Y, Zhang S, Darwin KH, Wang T, Li H. 2017. Mycobacterium tuberculosis proteasomal ATPase Mpa has a β-grasp domain that hinders docking with the proteasome core protease. Mol Microbiol 105(2):227–241.
Hsu HC, Singh PK, Fan H, Wang R, Sukenick G, Nathan C, Lin G, Li H. 2017. Structural basis for the species-selective binding of N,C-capped dipeptides to the Mycobacterium tuberculosis proteasome. Biochemistry 56(1):324–333.
Santos RLA, Bai L, Singh PK, Murakami N, Fan H, Zhan W, Zhu Y, Jiang X, Zhang K, Assker JP, Nathan CF, Li H, Azzi J, Lin G. 2017. Structure of human immunoproteasome with a reversible and noncompetitive inhibitor that selectively inhibits activated lymphocytes. Nat Commun 8(1):1692.
Hu K, Jastrab JB, Zhang S, Kovach A, Zhao G, Darwin KH, Li H. 2018. Proteasome substrate capture and gate opening by the accessory factor PafE from Mycobacterium tuberculosis. J Biol Chem 293(13):4713–4723.
Yu H, Lupoli TJ, Kovach A, Meng X, Zhao G, Nathan CF, Li H. 2018. ATP hydrolysis-coupled peptide translocation mechanism of Mycobacterium tuberculosis ClpB. Proc Natl Acad Sci U S A 115(41)::E9560–E9569.
Samanovic MI, Hsu HC, Jones MB, Jones V, McNeil MR, Becker SH, Jordan AT, Strnad M, Xu C, Jackson M, Li H, Darwin KH. 2018. Cytokinin signaling in Mycobacterium tuberculosis. MBio 19(3).
Zhan W, Hsu HC, Morgan T, Oullette T, Burns-Huang K, Hara R, Wright AG, Imaeda T, Okamoto R, Sato K, Michino M, Ramjee M, Aso K, Meinke P, Foley M, Nathan CR, Li H, Lin G. 2019. Selective phenylimidazole-based inhibitors of the Mycobacterium tuberculosis proteasome. J Med Chem.
Proteins can be modified with sugars either via an N-linkage on asparagine or via an O-linkage on the OH of a serine or threonine. N-glycosylation in eukaryotes is catalyzed by an ER embedded eight-protein membrane complex oligosaccharyltransferase (OST). Our earlier work mapped the subunit arrangement of the OST complex (Li et al 2008 Structure), and showed that OST binds to ribosome at a location near the nascent peptide exit tunnel (Harada et al 2009 Proc Natl Acad Sci U S A). Recently, we determined the atomic model of the OST, revealing the functions for many of the subunits and, unexpectedly, several phospholipids mediating subunit-subunit interaction in the transmembrane region (Bai et al 2018 Nature). Protein O-mannosyltransferase forms either a heterodimer or a homodimer, and associates with translocon in the ER membrane to perform co-translational protein O-mannosylation. We found the Pmt1-Pmt2 heterodimer has the GT-C fold and share structural homology with OST, confirming that protein O-mannosylation and protein N-glycosylation are evolutionarily related (Bai et al. 2019 NSMB).
O-glucosylation is a novel type of O-glycosylation. We recently studied enzymes that add a glucose-xylose-xylose trisaccharide on Notch receptor. We solved the structure of Rumi that adds the first glucose to Notch and found that Rumi recognizes a six-amino-acid sequence on many of the 36 EGF motifs of Notch and a hydrophobic patch (Yu et al 2016 Nat Chem Biol). Our study suggested that mutated forms of Rumi could be the cause of cancers because these mutant enzymes are unable to modify Notch. This observation links Rumi defects with cancers. XXYLT1 is a retaining glycosyltransferase that adds the third sugar of the trisaccharide — a xylose — to Notch. The chemical mechanism for retaining enzymes such as XXYLT1 had been controversial in the field for several decades. Our structural studies unambiguously supported the SNi-like retaining mechanism and disproved the double displacement mechanism (Yu et al 2015 Nat Chem Biol). We further solved the structure of an EGF motif covalently modified by a full O-glucose trisaccharide (Takeuchi et al 2017 J Biol Chem). The structure reveals that the glycan fills up a surface groove of the EGF with multiple contacts with the protein, providing a chemical basis for the stabilizing effects of the glycans.
Li H, Chavan M, Schindelin H, Lennarz WJ, Li H. 2008. Structure of the oligosaccharyl transferase complex at 12 Å resolution. Structure 16(3):432–440.
Harada Y, Li H, Li H, Lennarz WJ. 2009. Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site. Proc Natl Acad Sci U S A 106(17):6945–6949.
Yu H, Takeuchi M, LeBarron J, Kantharia J, London E, Bakker H, Haltiwanger RS, Li H*, Takeuchi H*. 2015. Notch-modifying xylosyltransferase structures support an SNi-like retaining mechanism. Nat Chem Biol 11(11):847–854.
Yu H, Takeuchi H, Takeuchi M, Liu Q, Kantharia J, Haltiwanger RS, Li H. 2016. Structural analysis of Notch-regulating Rumi reveals basis for pathogenic mutations. Nat Chem Biol 12(9):735–740.
Takeuchi H, Yu H, Hao H, Takeuchi M, Ito A, Li H, Haltiwanger RS. 2017. O-Glycosylation modulates the stability of epidermal growth factor-like repeats and thereby regulates Notch trafficking. J Biol Chem 292(38):15964–15973.
Bai L, Wang T, Zhao G, Kovach A, Li H. 2018. The atomic structure of a eukaryotic oligosaccharyltransferase complex. Nature 555(7696):328–333.
Bai L, Kovach A, You Q, Hsu C, Zhao G, Li H. 2019. Autoinhibition and activation mechanisms of the eurkaryotic lipid flippase Drs2p-Cdc50p. Nat Commun 10(1):4142.
Bai L, Kovach A, You Q, Kenny A, Li H. 2019. Structure of the eukaryotic protein O-mannosyltransferase Pmt1-Pmt2 complex. Nat Struct Mol Biol 26(8):704–711.