Huilin Li Laboratory

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; Feng et al. 2021, Nat Commun). In 2017, 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 holoenzyme structures of the leading strand DNA polymerase epsilon (Yuan et . 2020, Nat Commun), the lagging strand DNA polymerase delta (Zheng et al. 2020, Proc Natl Acad Sci U S A), and polymerase alpha-primase complex (Yuan et al. 2023, Nat Commun). We found Ctf4 trimer can coordinate two CMG helicases and one Pol alpha-primase into a replication factory core (Yuan et al. 2019, eLife).

Replicative polymerases are tethered to DNA by the PCNA sliding clamp. The PCNA is a topologically closed ring that needs to be cracked open to encircle DNA, and this is accomplished by the clamp loaders. We discovered that the canonical clamp loader RFC has evolved unique structural features to recognize both the 3′- and 5′-DNA ends to load PCNA onto gaps for lagging strand DNA synthesis and for DNA repair (Zheng et al. 2022, eLife). PCNA loading to the leading strand DNA is uniquely challenging because the 3’ DNA junction may be preoccupied by Polε recruited to DNA during origin activation. We have discovered how the yeast Ctf18-RFC binds to PCNA, DNA and Polε, leading to PCNA ring opening and lateral transfer of DNA from Polε to PCNA (Yuan et al. 2024, Science). We have also determined a series of PCNA loading intermediates of the human CTF18-RFC (He et al. 2024, Proc Natl Acad Sci U S A).

The PCNA ring needs to be taken off or unloaded from DNA upon completion of DNA synthesis. The PCNA unloading mechanism is least understood. We have discovered that both yeast Elg1-RFC and human ATAD5-RFC have evolved “locking loops” and a “plug domain” to prevent DNA binding, explaining why the Elg1/ATAD5-RFC are an exclusive PCNA unloader (Wang et al. 2024, Nat Struct Mol Biol).

In addition to PCNA, eukaryotes have evolved another DNA encircling ring called 9-1-1, which is a heterotrimer that functions in DNA damage checkpoint signaling. The 9-1-1 ring is loaded to the 5′ DNA by Rad24-RFC. We have demonstrated how Rad24-RFC loads the 9-1-1 checkpoint clamp ring onto the recessed 5′-end by binding a 5′-DNA at an external surface site and threading the 3′-ssDNA into the chamber and into 9-1-1 (Zheng et al. 2022, Nat Struct Mol Biol), how Rad24-RFC binds to a 10-nt and a 5-nt gap DNA, explaining why Rad24-RFC is unable to melt DNA ends and Rad24-RFC’s preference for a preexisting gap of >5 nt and suggesting a role of the 9-1-1 in gap repair (Zheng et al. 2023, Cell Rep).

Our work has advanced the field of eukaryotic DNA replication (Li et al. 2018, BioEssay; O’Donnell et al. 2018, Nat Struct Mol Biol; Yuan et al. 2020, Biochem J).

Related publications

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.

Feng X, Noguchi Y, Barbon M, Stillman B, Speck C, Li H. 2021. The structure of ORC-Cdc6 on an origin DNA reveals the mechanism of ORC activation by the replication initiator Cdc6. Nat Commun 12(1):3883.

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.

He Q, Wang F, Yao NY, O’Donnell ME*, Li H*. 2024. Structures of the human leading strand Polε–PCNA holoenzyme. Nat Commun 15(7847).
*Co-corresponding authors

He Q, Wang F, O’Donnell ME, Li H. 2024. Cryo-EM reveals a nearly complete PCNA loading process and unique features of the human alternative clamp loader CTF18-RFC. Proc Nat Acad Sci U S A 121(18):e2319727121.

Li H, O’Donnell ME. 2018. The eukaryotic CMG helicase at the replication fork: Emerging architecture reveals an unexpected mechanism. BioEssays 40(3).

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.

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.

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.

Wang F, He Q, Yao NY, O’Donnell ME, Li H. 2024. The human ATAD5 has evolved unique structural elements to function exclusively as a PCNA unloader. Nat Struct Mol Biol.

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.

Yuan Z, Georgescu R, Yao NY, Yurieva O, O’Donnell ME, Li H. 2024. Mechanism of PCNA loading by Ctf18-RFC for leading-strand DNA synthesis. Science 385(6708).

Yuan Z, Georgescu R, Li H*, O’Donnell ME*. 2023. Molecular choreography of primer synthesis by the eukaryotic Pol α-primase. Nat Commun 14(1):3697.
*Co-corresponding authors

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.

Yuan Z, Georgescu R, Schauer GD, O’Donnell ME, Li H. 2020. Structure of the polymerase ε holoenzyme and atomic model of the leading strand replisome. Nat Commun11(1):3156.

Yuan Z, Li H. 2020. Molecular mechanisms of eukaryotic origin initiation, replication fork progression, and chromatin maintenance. Biochem J 477(18):3499–3525.

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.

Zheng F, Georgescu RE, Li H, O’Donnell ME. 2020. Structure of eukaryotic DNA polymerase δ bound to the PCNA clamp while encircling DNA. Proc Natl Acad Sci U S A117(48):30344–30353.

Zheng F, Georgescu R, Yao NY, Li H, O’Donnell ME. 2022. Cryo-EM structures reveal that RFC recognizes both the 3′- and 5′-DNA ends to load PCNA onto gaps for DNA repair. eLife 11:e77469.

Zheng F, Yao NY, Georgescu RE, Li H*, O’Donnell ME*. 2024. Structure of the PCNA unloader Elg1-RFC. Sci Adv 10(9).
*Co-corresponding authors

Zheng F, Georgescu RE, Yao NY, O’Donnell ME, Li H. 2023. Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification on biology. Cell Rep 42(7):112694.

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, Nat Struct Mol Biol).

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.

The core of heparin sulfate is a copolymer of two alternating sugars GlcA and GlcNAc. This copolymer core is synthesized by exostosin-1 and exostosin-2, which form an obligatory heterodimer (EXT1-2). By solving a series structures of the enzyme complexed with donors and acceptors, we have recently shown that each subunit contains one activity: the EXT1 GT-B domain contains the β1,4GlcA transferase domain and the EXT2 GT-A domain the α1,4GlcNAc transferase domain (Li et al. 2023, Nat Chem Biol). Because the two catalytic sites are far apart (90 Å), we suggested that HS is synthesized by a dissociative process.

Glycans on proteins can be further modified for new functions. For example, vertebrates have evolved the mannose 6-phosphate (M6P) recognition system to deliver lysosomal hydrolases to lysosomes. In this system, a phosphotransferase (PTase, N-acetylglucosamine (GlcNAc)-1-phosphotransferase) adds GlcNAc-phosphate to mannose residues in the N-glycan of the lysosomal hydrolases. We have determined the structure of the human PTase and discovered a hockey stick-like motif that controls activation of the enzyme (Li et al. 2022, Nat Struct Mol Biol). More recently, we found that a highly truncated human PTase is hyperactive because the truncation has removed the hockey stick, making the enzyme in a constitutively active state (Li et al. 2024, J Biol Chem). Interestingly, despite the truncated PTase resembles the simpler PTase homolog in fruit fly, the fly enzyme does not act on lysosomal hydrolases (Li et al. 2024, J Biol Chem).

Concomitant to their glycosylation, nascent membrane proteins are inserted into the lipid bilayer with the help of membrane chaperones such as the ER membrane complex whose structure was determined by us recently (Bai et al. 2020, Nature; Bai et al. 2023, Curr Opin Struct Biol). Then, the glycosylated and folded membrane proteins are trafficked from ER through Golgi to reach cell surface membrane. This transport process is facilitated by several lipid flippases. We have determined the structure and substrate binding and flipping mechanisms for three yeast lipid flippases: Drs2-Cdc50 (Bai et al. 2019, Nat Commun), Neo1 (Bai et al. 2021, Nat Commun), and Dnf1/Dnf2-Cdc50 (Bai et al. 2020, eLife). This work has improved our understanding of molecular mechanisms of flipping lipids from outer leaflet to the inner leaflet of the membrane bilayers (Duan et al. 2024, J Biol Chem). To further understand how the Drs2-Cdc50 flippase is activated by Arl1-Gea2 complex, we have recently revealed that Gea2 assembles a stable dimer, and unexpectedly, Arl1 binds to the DCB rather than the SEC7 domain (Duan et al. 2024, Nat Commun).

Related Publications

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.

Bai L, You Q, Feng X, Kovach A, Li H. 2020. Structure of the ER membrane complex, a transmembrane-domain insertase. Nature 584:475–478.

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, You Q, Jain BK, Duan HD, Kovach A, Graham TR, Li H. 2020. Transport mechanism of P4 ATPase phosphatidylcholine flippases. eLife 9:e62163.

Bai L, Jain BK, You Q, Duan HD, Takar M, Graham TR, Li H. 2021. Structural basis of the P4B ATPase lipid flippase activity. Nat Commun 12:5963.

Bai L, Li H. 2023. Structural insights into the membrane chaperones for multi-pass membrane protein biogenesis. Curr Opin Struct Biol 79:102563.

Duan HD, Li H. 2024. Consensus, controversies, and conundrums of P4-ATPases: the emerging face of eukaryotic lipid flippases. J Biol Chem 300(6):107387.

Duan HD, Jain BK, Li H, Graham TR*, Li H*. 2024. Structural insight into an Arl1-ArfGEF complex involved in Golgi recruitment of a GRIP-domain golgin. Nat Commun 15:1942.
*Co-corresponding authors

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.

Li H, Chapla D, Amos RA, Ramiah A, Moremen KW, Li H. 2023. Structural basis for heparan sulfate co-polymerase action by the EXT1–2 complex. Nat Chem Biol.

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.

Li H, Doray B, Jennings BC, Lee W-S, Liu L, Kornfeld S, Li H. 2024. Structure of a truncated human GlcNAc-1-phosphotransferase variant reveals the basis for its hyperactivity. J Biol Chem 300(9):107706.

Li H, Lee WS, Feng X, Bai L, Jennings BC, Liu L, Doray B, Canfield WM, Kornfeld S, Li H. 2022. Structure of the human GlcNAc-1-phosphotransferase αβ subunits reveals regulatory mechanism for lysosomal enzyme glycan phosphorylation. Nat Struct Molec Biol 29(4):348–356.

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.

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.

Tuberculosis (TB) kills 1.5 million to 2 million people globally every year. In the U.S. alone, 13 million people have latent TB infection; and about 10% 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 mechanisms (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; Yin et al. 2021, J Biol Chem), 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 and macrocyclic peptide-based inhibitors (Zhan et al. 2019, J Med Chem; Zhang et al. 2021, J Med Chem). Remarkably, some of the dipeptide variants exhibited potent and selective inhibition to the human immunoproteasome (Santos et al. 2017, Nat Commun).

In addition to removing the damaged proteins by the Pup-proteasome system, Mtb also uses the DnaK-ClpB bi-chaperone system to rescue and refold damaged proteins. We have studied the protein disaggregation mechanism by the Mtb ClpB hexamer (Yu et al. 2018, Proc Natl Acad Sci U S A) and revealed how ClpB and DnaK work together to synergistically disaggregate and refold protein aggregates (Yin et al. 2021, Cell Rep), and discovered that the GrpE dimer facilitates not only the nucleotide exchange but also the protein substrate release from the DnaK (Xiao et al. 2024, Nat Commun).

Related Publications

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

Xiao X, Fay A, Santos Molina P, Kovach A, Glickman MS, Li H. 2024. Structure of the M. tuberculosis DnaK−GrpE complex reveals how key DnaK roles are controlled. Nat Comm 15:660.

Yin Y, Kovach A, Hsu HC, Darwin KH, Li H. 2021. The mycobacterial proteasomal ATPase Mpa forms a gapped ring to engage the 20S proteasome. J Biol Chem 100713.

Yin Y, Feng X, Yu H, Fay A, Kovach A, Glickman MS, Li H. 2021. Structural basis for aggregate dissolution and refolding by the Mycobacterium tuberculosis ClpB-DnaK bi-chaperone system. Cell Rep 35(8):109166.

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.

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 62(20):9246–9253.

Zhang H, Hsu HC, Kahne SC, Hara R, Zhan W, Jiang X, Burns-Huang K, Ouellette T, Imaeda T, Okamoto R, Kawasaki M, Michino M, Wong TT, Toita A, Yukawa T, Moraca F, Vendome J, Saha P, Sato K, Aso K, Ginn J, Meinke PT, Foley M, Nathan CF, Darwin KH, Li H, Lin G. 2021. Macrocyclic peptides that selectively inhibit the Mycobacterium tuberculosis proteasome. J Med Chem 64(9):6262–6272.

Energy conservation in modern-day mitochondria relies on oxygen as the ultimate electron acceptor. However, life evolved 4 billion years ago, and atmospheric oxygen was available only 2.5 billion years ago. In the absence of oxygen, ancestral life has used proton or elemental sulfur as electron acceptors, mediated by the ancestral respiratory complexes such as the membrane-bound hydrogenase (MBH) and membrane-bound sulfane sulfur reductase (MBS), which now exist in hyperthermophile Pyrococcus furiosus. We have recently solved the structures of MBH (Yu et al. 2018, Cell) and MBS (Yu et al. 2021, Nat Commun), shedding light on their redox-coupled ion pumping mechanisms and their evolutionary relationships with the modern-day respiratory Complex I (Yu et al. 2021, JBC review).

Electron bifurcation was first discovered by Peter Mitchell in respiratory Complex III and is quinone-based. Recently, flavin-based electron bifurcation (FBEB) is found to be a widespread energy conservation mechanism in microbial metabolism. These oxidoreductase-type enzymes catalyze endergonic reduction of low-potential, high-energy substrates by simultaneously coupling them to the reduction of high-potential, low-energy substrates in an exergonic reaction. Electron bifurcation is therefore used by microorganisms as a general mechanism to produce low-potential, high-energy electrons without the expenditure of ATP. In collaboration with the Michael Adams lab, we have recently solved the structures of the membrane-associated Fix/EtfABCX (Feng et al. 2021, Proc Natl Acad Sci U S A) and the NiFe-HydABCSL (Feng et al. 2022, Sci Adv, in print), revealing their electron transport pathways and potential bifurcation mechanisms.

Related Publications

Feng X, Schut G, Lipscomb G, Li H, Adams M. 2021. Cryoelectron microscopy structure and mechanism of the membrane-associated electron-bifurcating flavoprotein Fix/EtfABCX. Proc Natl Acad Sci U S A 118(2).

Feng X, Schut G, Haja D, Adama M, Li H. 2022. Structure and electron transfer pathways of an electron bifurcating NiFe-hydrogenase. Sci Adv.

Yu H, Wu CH, Schut G, Haja D, Zhao G, Peters J, Adams M, Li H. 2018. Structure of an ancient respiratory system. Cell 173(7):1636-1649.

Yu H, Haja DK, Schut GJ, Wu CH, Meng X, Zhao G, Li H, Adams MWW. 2020. Structure of the respiratory MBS complex reveals iron-sulfur cluster catalyzed sulfane sulfur reduction in ancient life. Nat Commun 11(1):5953.

Yu H, Schut G, Haja D, Adams M, Li H. 2021. Evolution of complex I-like respiratory complexes. J Biol Chem 296: 100740. (Invited review)