Huilin Li

Stony Brook University, Stony Brook, New York, United States

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Publications (58)633.97 Total impact

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    ABSTRACT: The chaperone-usher (CU) pathway assembles organelles termed pili or fimbriae in Gram-negative bacteria. Type 1 pili expressed by uropathogenic Escherichia coli are prototypical structures assembled by the CU pathway. Biogenesis of pili by the CU pathway requires a periplasmic chaperone and an outer-membrane protein termed the usher (FimD). We show that the FimD C-terminal domains provide the high-affinity substrate-binding site but that these domains are masked in the resting usher. Domain masking requires the FimD plug domain, which serves as a switch controlling usher activation. We demonstrate that usher molecules can act in trans for pilus biogenesis, providing conclusive evidence for a functional usher oligomer. These results reveal mechanisms by which molecular machines such as the usher regulate and harness protein-protein interactions and suggest that ushers may interact in a cooperative manner during pilus assembly in bacteria.
    Nature Structural & Molecular Biology 06/2015; DOI:10.1038/nsmb.3044 · 11.63 Impact Factor
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    ABSTRACT: Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and protein degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world's most devastating pathogens.
    Proceedings of the National Academy of Sciences 03/2015; 112(14). DOI:10.1073/pnas.1423319112 · 9.81 Impact Factor
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    ABSTRACT: Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2-7 (minichromosome maintenance proteins 2-7) double hexamer. During S phase, each Mcm2-7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC-Cdc6 function to recruit a single Cdt1-Mcm2-7 heptamer to replication origins prior to Cdt1 release and ORC-Cdc6-Mcm2-7 complex formation, but how the second Mcm2-7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC-Cdc6-Mcm2-7 complex and an ORC-Cdc6-Mcm2-7-Mcm2-7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2-7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2-7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.
    Genes & Development 10/2014; 28(20):2291-303. DOI:10.1101/gad.242313.114 · 12.64 Impact Factor
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    ABSTRACT: The regulated loading of the replicative helicase minichromosome maintenance proteins 2–7 (MCM2–7) onto replication origins is a prerequisite for replication fork establishment and genomic stability. Origin recognition complex (ORC), Cdc6, and Cdt1 assemble two MCM2–7 hexamers into one double hexamer around dsDNA. Although the MCM2–7 hexamer can adopt a ring shape with a gap between Mcm2 and Mcm5, it is unknown which Mcm interface functions as the DNA entry gate during regulated helicase loading. Here, we establish that the Saccharomyces cerevisiae MCM2–7 hexamer assumes a closed ring structure, suggesting that helicase loading requires active ring opening. Using a chemical biology approach, we show that ORC–Cdc6–Cdt1-dependent helicase loading occurs through a unique DNA entry gate comprised of the Mcm2 and Mcm5 subunits. Controlled inhibition of DNA insertion triggers ATPase-driven complex disassembly in vitro, while in vivo analysis establishes that Mcm2/Mcm5 gate opening is essential for both helicase loading onto chromatin and cell cycle progression. Importantly, we demonstrate that the MCM2–7 helicase becomes loaded onto DNA as a single hexamer during ORC/Cdc6/Cdt1/MCM2–7 complex formation prior to MCM2–7 double hexamer formation. Our study establishes the existence of a unique DNA entry gate for regulated helicase loading, revealing key mechanisms in helicase loading, which has important implications for helicase activation.
    Genes & Development 08/2014; 28:1653-1666. DOI:10.1101/gad.242404.114 · 12.64 Impact Factor
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    ABSTRACT: Phytochromes are multidomain photoswitches that drive light perception in plants and microorganisms by coupling photoreversible isomerization of their bilin chromophore to various signaling cascades. How changes in bilin conformation affect output by these photoreceptors remains poorly resolved and might include several species-specific routes. Here, we present detailed three-dimensional models of the photosensing module (PSM), and a picture of an entire dimeric photoreceptor through structural analysis of the Deinococcus radiodurans phytochrome BphP assembled with biliverdin (BV). A 1.16 angstrom-resolution crystal structure of the bilin-binding pocket as Pr illuminated the intricate network of bilin/protein/water interactions and confirmed the protonation and ZZZssa conformation of BV. Structural and spectroscopic comparisons with the photochemically-compromised D207A mutant revealed that substitutions of Asp207 allow inclusion of cyclic porphyrins in addition to BV. A crystal structure of the entire PSM showed a head-to-head, twisted dimeric arrangement with bowed helical spines and a hairpin protrusion connecting the cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) and phytochrome-specific (PHY) domains. A key conserved hairpin feature is its anti-parallel, two β-strand stem, which we show by mutagenesis to be critical for BphP photochemistry. Single-particle electron microscopic images of the full-length BphP dimer in the dark-adapted Pr and photoactivated Pfr states revealed a large-scale reorientation of the PHY domain relative to the GAF domain, which alters the position of the downstream histidine kinase output module. Together, our data support a model whereby bilin photoisomerization alters GAF/PHY domain interactions through conformational modification of the hairpin, which regulates signaling by impacting the relationship between sister output modules.
    Journal of Biological Chemistry 07/2014; 289(35). DOI:10.1074/jbc.M114.571661 · 4.60 Impact Factor
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    ABSTRACT: In addition to binding intracellular fatty acids, fatty-acid-binding proteins (FABPs) have recently been reported to also transport the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), arachidonic acid derivatives that function as neurotransmitters and mediate a diverse set of physiological and psychological processes. To understand how the endocannabinoids bind to FABPs, the crystal structures of FABP5 in complex with AEA, 2-AG and the inhibitor BMS-309403 were determined. These ligands are shown to interact primarily with the substrate-binding pocket via hydrophobic interactions as well as a common hydrogen bond to the Tyr131 residue. This work advances our understanding of FABP5-endocannabinoid interactions and may be useful for future efforts in the development of small-molecule inhibitors to raise endocannabinoid levels.
    Acta Crystallographica Section D Biological Crystallography 02/2014; 70(Pt 2):290-298. DOI:10.1107/S1399004713026795 · 7.23 Impact Factor
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    ABSTRACT: Our primary goal has been to understand how chromosomes are duplicated during the cell division cycle to ensure faithful inheritance of genetic information in eukaryotes. By initially investigating SV40 DNA replication, we reconstituted the entire process of DNA replication from the SV40 origin and in so doing discovered how the replication fork is assembled. Later, from studies on replication of DNA from chromosomal origins of DNA replication in eukaryotic cells, we discovered an ATP-dependent protein machine, the Origin Recognition Complex (ORC) that is required to form a pre-Replicative Complex (pre-RC) at all origins of DNA replication prior to S phase. The process of pre-RC assembly at origins of DNA replication licenses chromosomes for subsequent DNA replication during the S phase of the cell cycle. Pre-RC assembly has been reconstituted in vitro with purified proteins and structural studies have revealed a conserved mechanism of protein assembly on origin DNA. Activation of DNA replication requires multiple protein kinase signaling systems, including the Cyclin-Dependent Protein Kinase (CDK), the Cdc7-Dbf4 Protein Kinase (DDK) and the Mec1 (ATM/ATR) checkpoint kinase
    The FASEB Journal 01/2014; 28:27-1. · 5.48 Impact Factor
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    ABSTRACT: Mycobacterium tuberculosis (Mtb) restrains immune responses well enough to escape eradication but elicits enough immunopathology to ensure its transmission. Here we provide evidence that this host-pathogen relationship is regulated in part by a cytosolic, membrane-associated protein with a unique structural fold, encoded by the Mtb gene rv0431. The protein acts by regulating the quantity of Mtb-derived membrane vesicles bearing Toll-like receptor 2 ligands, including the lipoproteins LpqH and SodC. We propose that rv0431 be named "vesiculogenesis and immune response regulator."
    Proceedings of the National Academy of Sciences 11/2013; 110(49). DOI:10.1073/pnas.1320118110 · 9.81 Impact Factor
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    Cell cycle (Georgetown, Tex.) 08/2013; 12(18). DOI:10.4161/cc.26132 · 5.01 Impact Factor
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    ABSTRACT: In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC-Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using purified components in the presence of ATP-γS, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the ORC-Cdc6 and Cdt1-MCM2-7 heteroheptamers called the OCCM. Cryo-EM studies of this 14-subunit complex reveal that the two separate heptameric complexes are engaged extensively, with the ORC-Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer. The conformation of ORC-Cdc6 undergoes a concerted change into a right-handed spiral with helical symmetry that is identical to that of the DNA double helix. The resulting ORC-Cdc6 helicase loader shows a notable structural similarity to the replication factor C clamp loader, suggesting a conserved mechanism of action.
    Nature Structural & Molecular Biology 07/2013; DOI:10.1038/nsmb.2629 · 11.63 Impact Factor
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    Journal of Molecular Biology 03/2013; 425(5):968. DOI:10.1016/j.jmb.2012.12.020 · 3.96 Impact Factor
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    ABSTRACT: Proteasomes are ATP-dependent protein degradation machines present in all archaea and eukaryotes, and found in several bacterial species of the order Actinomycetales. Mycobacterium tuberculosis (Mtb), an Actinomycete pathogenic to humans, requires proteasome function to cause disease. In this chapter, we describe what is currently understood about the biochemistry of the Mtb proteasome and its role in virulence. The characterization of the Mtb proteasome has led to the discovery that proteins can be targeted for degradation by a small protein modifier in bacteria as they are in eukaryotes. Furthermore, the understanding of proteasome function in Mtb has helped reveal new insight into how the host battles infections.
    Sub-cellular biochemistry 01/2013; 66:267-95. DOI:10.1007/978-94-007-5940-4_10
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    ABSTRACT: Many membrane proteins exist and function as oligomers, but how monomers oligomerize in the cell membrane remains poorly understood. AcrB is an obligate homo-trimer. We previously found that the folding of individual subunit precedes oligomerization. Following folding, individual AcrB subunits must locate and interact with each other in order to dimerize and eventually trimerize. It has been unclear if AcrB trimerization is a spontaneous process following the "chance encounter and random assembling" mechanism. In other words, it is currently unknown whether monomeric subunits diffuse freely to "search" for each other after they are co-translationally inserted and folded into the cell membrane. Using four sets of experiments exploiting AcrB variants with different fusion tags, disulfide trapping, and activity measurement, here we showed that AcrB variants co-expressed in the same Escherichia coli cell did co-assemble into hybrid trimers in vivo. However, the level of co-assembly measured experimentally was not consistent with calculations derived from random assembling. The potential role of the polysome structure during protein translation and the resultant clustering effect were discussed as a potential explanation for the observed bias in AcrB subunit assembling in vivo. Our results provide new insights into the dynamic assembling and equilibration process of obligate homo-oligomeric membrane proteins in the cell membrane.
    Journal of Molecular Biology 07/2012; 423(1):123-34. DOI:10.1016/j.jmb.2012.06.036 · 3.96 Impact Factor
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    ABSTRACT: Papaya mosaic virus (PapMV) is a filamentous plant virus that belongs to the Alphaflexiviridae family. Flexible filamentous viruses have defied more than two decades of effort in fiber diffraction, and no high-resolution structure is available for any member of the Alphaflexiviridae family. Here, we report our structural characterization of PapMV by X-ray crystallography and cryo-electron microscopy three-dimensional reconstruction. We found that PapMV is 135Å in diameter with a helical symmetry of ~10 subunits per turn. Crystal structure of the C-terminal truncated PapMV coat protein (CP) reveals a novel all-helix fold with seven α-helices. Thus, the PapMVCP structure is different from the four-helix-bundle fold of tobacco mosaic virus in which helix bundling dominates the subunit interface in tobacco mosaic virus and conveys rigidity to the rod virus. PapMV CP was crystallized as an asymmetrical dimer in which one protein lassoes the other by the N-terminal peptide. Mutation of residues critical to the inter-subunit lasso interaction abolishes CP polymerization. The crystal structure suggests that PapMV may polymerize via the consecutive N-terminal loop lassoing mechanism. The structure of PapMV will be useful for rational design and engineering of the PapMV nanoparticles into innovative vaccines.
    Journal of Molecular Biology 05/2012; 422(2):263-73. DOI:10.1016/j.jmb.2012.05.032 · 3.96 Impact Factor
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    ABSTRACT: The eukaryotic origin recognition complex (ORC) interacts with and remodels origins of DNA replication prior to initiation in S phase. Here, we report a single-particle cryo-EM-derived structure of the supramolecular assembly comprising Saccharomyces cerevisiae ORC, the replication initiation factor Cdc6, and double-stranded ARS1 origin DNA in the presence of ATPγS. The six subunits of ORC are arranged as Orc1:Orc4:Orc5:Orc2:Orc3, with Orc6 binding to Orc2. Cdc6 binding changes the conformation of ORC, in particular reorienting the Orc1 N-terminal BAH domain. Segmentation of the 3D map of ORC-Cdc6 on DNA and docking with the crystal structure of the homologous archaeal Orc1/Cdc6 protein suggest an origin DNA binding model in which the DNA tracks along the interior surface of the crescent-like ORC. Thus, ORC bends and wraps the DNA. This model is consistent with the observation that binding of a single Cdc6 extends the ORC footprint on origin DNA from both ends.
    Structure 03/2012; 20(3):534-44. DOI:10.1016/j.str.2012.01.011 · 6.79 Impact Factor
  • Huilin Li, Bruce Stillman
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    ABSTRACT: The origin recognition complex (ORC) was first discovered in the baker's yeast in 1992. Identification of ORC opened up a path for subsequent molecular level investigations on how eukaryotic cells initiate and control genome duplication each cell cycle. Twenty years after the first biochemical isolation, ORC is now taking on a three-dimensional shape, although a very blurry shape at the moment, thanks to the recent electron microscopy and image reconstruction efforts. In this chapter, we outline the current biochemical knowledge about ORC from several eukaryotic systems, with emphasis on the most recent structural and biochemical studies. Despite many species-specific properties, an emerging consensus is that ORC is an ATP-dependent machine that recruits other key proteins to form pre-replicative complexes (pre-RCs) at many origins of DNA replication, enabling the subsequent initiation of DNA replication in S phase.
    Sub-cellular biochemistry 01/2012; 62:37-58. DOI:10.1007/978-94-007-4572-8_3
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    ABSTRACT: Type 1 pili are the archetypal representative of a widespread class of adhesive multisubunit fibres in Gram-negative bacteria. During pilus assembly, subunits dock as chaperone-bound complexes to an usher, which catalyses their polymerization and mediates pilus translocation across the outer membrane. Here we report the crystal structure of the full-length FimD usher bound to the FimC-FimH chaperone-adhesin complex and that of the unbound form of the FimD translocation domain. The FimD-FimC-FimH structure shows FimH inserted inside the FimD 24-stranded β-barrel translocation channel. FimC-FimH is held in place through interactions with the two carboxy-terminal periplasmic domains of FimD, a binding mode confirmed in solution by electron paramagnetic resonance spectroscopy. To accommodate FimH, the usher plug domain is displaced from the barrel lumen to the periplasm, concomitant with a marked conformational change in the β-barrel. The amino-terminal domain of FimD is observed in an ideal position to catalyse incorporation of a newly recruited chaperone-subunit complex. The FimD-FimC-FimH structure provides unique insights into the pilus subunit incorporation cycle, and captures the first view of a protein transporter in the act of secreting its cognate substrate.
    Nature 06/2011; 474(7349):49-53. DOI:10.1038/nature10109 · 42.35 Impact Factor
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    ABSTRACT: In Saccharomyces cerevisiae, some of the nascent chains can be post-translationally translocated into the endoplasmic reticulum through the heptameric post-translational translocon complex (post-translocon). This membrane-protein complex is composed of the protein-conducting channel and the tetrameric Sec62/63 complex. The Sec62/63 complex plays crucial roles in targeting of the signal recognition particle-independent protein substrate to the protein-conducting channel and in assembly of the post-translocon. Although the molecular mechanism of the post-translational translocation process has been well established, the structure of the post-translocon and how the channel and the Sec62/63 complex form the heptameric complex are largely uncharacterized. Here, we report a 20-Å resolution cryo-electron microscopy structure of the post-translocon. The purified post-translocon was found to have a mass of 287 kDa, which is consistent with the unit stoichiometry of the seven subunits as determined by a cysteine labeling experiment. We demonstrated that Triton X-100 dissociated the heptameric complex into three subcomplexes identified as the trimeric translocon Sec61-Sbh1-Sss1, the Sec63-Sec71-Sec72 trimer, and the heterotetramer Sec62-Sec63-Sec71-Sec72, respectively. Additionally, a role of the sixth cytosolic loop of Sec61 in assembly of the post-translocon was demonstrated. Mutations of conserved, positively charged amino acid residues in the loop caused decreased formation of the post-translocon. These studies provide the first architectural description of the yeast post-translocon.
    Journal of Biological Chemistry 01/2011; 286(4):2956-65. DOI:10.1074/jbc.M110.159517 · 4.60 Impact Factor

Publication Stats

3k Citations
633.97 Total Impact Points

Institutions

  • 2008–2015
    • Stony Brook University
      • Department of Biochemistry and Cell Biology
      Stony Brook, New York, United States
  • 2004–2014
    • Brookhaven National Laboratory
      • Biology Department
      New York, New York, United States
  • 2013
    • MRC Clinical Sciences Centre
      London Borough of Harrow, England, United Kingdom
    • CUNY Graduate Center
      New York, New York, United States
  • 2008–2012
    • Cold Spring Harbor Laboratory
      Cold Spring Harbor, New York, United States
  • 2007
    • Lehigh University
      • Department of Chemistry
      Bethlehem, Pennsylvania, United States
    • Duke University Medical Center
      Durham, North Carolina, United States
  • 2002
    • Brandeis University
      • Department of Biology
      Волтам, Massachusetts, United States
  • 2001
    • Lawrence Berkeley National Laboratory
      • Life Sciences Division
      Berkeley, CA, United States