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Scholarly Works (3 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2021)

Plants and animals respond to pathogen invasion via intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) that directly interact with pathogen proteins or indirectly detect pathogen-derived alterations of the host proteome. Upon recognition of pathogen invasion, NLRs trigger an immune response that resolves in a variety of ways depending on the type of NLR being activated. The overall architecture of NLRs is highly conserved, consisting of an C-terminal LRR platform that determines substrate specificity and a central nucleotide-binding oligomerization domain. The N-terminal domain varies among NLRs and determines the mechanism utilized by the host to activate the immune response. In plants, NLRs have been classified according to their N-terminal domains: TNLs (TIR-NLRs), CNLs (CC-NLRs) and RNLs (CCR-NLRs). Pathogen detection and oligomerization of the NLR activates these N-terminal domains by bringing them in close-contact. In all 3 cases, association of the N-terminal domain leads to localized cell death and expression of disease resistance. While the TIR domains of TNLs have been shown to possess oligomerization-dependent NADase activity, it is not understood how their association leads to activation.

The structure of the ROQ1-XopQ complex, a TNL immune receptor bound to its pathogen substrate, was used as a model to study the mechanism of direct effector recognition, oligomerization, and TIR domain activation. ROQ1 has been shown to physically interact with the Xanthomonas effector XopQ, causing it to oligomerize and trigger a TIR-dependent hypersensitive cell death response. We co-expressed, extracted and purified the assembled ROQ1-XopQ complex from ROQ1’s native host, Nicotiana benthamiana, and solved its structure by cryo-EM to 3.8 Å resolution. The interactions described in our structure were further confirmed by in vivo mutational analysis.

Our structure reveals that ROQ1 forms a tetrameric resistosome upon recognizing XopQ. The LRR and a post-LRR domain, termed C-JID (C-terminal Jelly-roll/Ig-like Domain), form a horseshoe shaped scaffold that curls around the pathogen effector, thereby recognizing multiple regions of the substrate. Roq1 binds to XopQ via surface exposed residues that make up the LRR scaffold as well as an elongated loop between two leucine-rich repeats that forms a small amphipathic α-helix at the site of interaction. The mode of substrate recognition utilized by the C-JID is reminiscent of the way immunoglobulins bind to their antigen. Similarly to the complementary-determining regions of antibodies, interconnecting loops emerging from the C-JID β-sandwich structure make substrate-specific contacts with XopQ. In particular, an extended loop of the C-JID dives into the active site cleft of XopQ and interacts with conserved residues required for nucleoside binding, suggesting that ROQ1 not only recognizes its substrate but also inhibits its ligand-binding function. The NB-ARC domain (NBD, HD1, WHD) of ROQ1 responsible for oligomerization is found in an ATP-bound state. Individual protomers intercalate in a similar fashion as found in other NLR structures, promoting association between the N-terminal TIR domains. The TIR domains bind to each other via two distinct interfaces (termed AE and BE) causing them to form a dimer of dimers. BE-interface contacts cause a conformational rearrangement in a loop, termed the BB-loop, at the periphery of the TIR domain active site that exposes the putative catalytic glutamate, suggested to participate in NAD+ cleavage. These results provide a rationale for the previously determined oligomerization-dependence of TIR domain NADase activity.

A step-by-step mechanism for ROQ1 immune signaling is proposed based on our structure of the activated complex and previous biochemical studies. The LRR and C-JID of ROQ1 recognize the pathogen effector via direct contacts with its surface and active site residues. Detection of the substrate releases autoinhibitory contacts between the NB-ARC domain and the LRR allowing the NB-ARC domain to transition to an ATP-bound oligomerization prone state. Complex assembly brings the TIR domains in close contact leading to opening of the NADase active site in an interface-dependent manner.

    Cover page: Structure of the activated ROQ1 resistosome directly recognizing the pathogen effector XopQ
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2016)
    Challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. One such process is chaperone-assisted protein folding. Obtaining structural ensembles of chaperone-substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. To address this problem, we devised a new structural biology approach based on X-ray crystallography, termed residual electron and anomalous density (READ). READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the Escherichia coli chaperone Spy, and to capture a series of snapshots depicting the various folding states of Im7 bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded to native-like states and reveals how a substrate can explore its folding landscape while being bound to a chaperone.
      Cover page: Visualizing chaperone-assisted protein folding
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2024)
      Innate immune responses to microbial pathogens are regulated by intracellular receptors known as nucleotide-binding leucine-rich repeat receptors (NLRs) in both the plant and animal kingdoms. Across plant innate immune systems, "helper" NLRs (hNLRs) work in coordination with "sensor" NLRs (sNLRs) to modulate disease resistance signaling pathways. Activation mechanisms of hNLRs based on structures are unknown. Our research reveals that the hNLR, known as NLR required for cell death 4 (NRC4), assembles into a hexameric resistosome upon activation by the sNLR Bs2 and the pathogenic effector AvrBs2. This conformational change triggers immune responses by facilitating the influx of calcium ions (Ca2+) into the cytosol. The activation mimic alleles of NRC2, NRC3, or NRC4 alone did not induce Ca2+ influx and cell death in animal cells, suggesting that unknown plant-specific factors regulate NRCs' activation in plants. These findings significantly advance our understanding of the regulatory mechanisms governing plant immune responses.
        Cover page: Activation of the helper NRC4 immune receptor forms a hexameric resistosome
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