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Open Access Publications from the University of California

Scholarly Works (9 results)

  • Article
  • Peer Reviewed
UC San Francisco Previously Published Works (2014)
Cytoskeletal filaments form diverse superstructures that are highly adapted for specific functions. The recently discovered TubZ subfamily of tubulins is involved in type III plasmid partitioning systems, facilitating faithful segregation of low copy-number plasmids during bacterial cell division. One such protein, TubZ-Bt, is found on the large pBtoxis plasmid in Bacillus thuringiensis, and interacts via its extended C terminus with a DNA adaptor protein TubR. Here, we use cryo-electron microscopy to determine the structure of TubZ-Bt filaments and light scattering to explore their mechanism of polymerization. Surprisingly, we find that the helical filament architecture is remarkably sensitive to nucleotide state, changing from two-stranded to four-stranded depending on the ability of TubZ-Bt to hydrolyze GTP. We present pseudoatomic models of both the two- and four-protofilament forms based on cryo-electron microscopy reconstructions (10.8 Å and 6.9 Å, respectively) of filaments formed under different nucleotide states. These data lead to a model in which the two-stranded filament is a necessary intermediate along the pathway to formation of the four-stranded filament. Such nucleotide-directed structural polymorphism is to our knowledge an unprecedented mechanism for the formation of polar filaments.
    Cover page: Bacterial tubulin TubZ-Bt transitions between a two-stranded intermediate and a four-stranded filament upon GTP hydrolysis
    • Thesis
    • Peer Reviewed
    UC San Francisco Electronic Theses and Dissertations (2013)

    The tubulin superfamily is a unique set of GTPases, which, while they have high sequence diversity, have remarkably similar tertiary structures. These proteins share a very conserved longitudinal interface, but are able to form a surprisingly wide variety of super structures within both eukaryotic and prokaryotic cells — from the familiar 13-protofilament microtubule assembled from αβ–tubulin heterodimers to the single protofilaments of the ubiquitous prokaryotic FtsZ seen in vitro. Additionally in eukaryotes, monomeric tubulin family members have the ability to affect the assembly of filaments, such as nucleation of microtubules with γ–tubulin, or involvement in the assembly of B– and C– microtubules in the centriole microtubule triplets (ε– and δ–tubulin respectively). Each tubulin family member, both prokaryotic and eukaryotic is exquisitely tuned to perform its cellular function, with a unique superstructure and dynamics.

    The main focus of this manuscript is a structural and biochemical investigation of a recently discovered bacterial tubulin TubZ–Bt. TubZ–Bt is located on the pBtoxis virulence plasmid in Bacillus thuringiensis, and is necessary to maintain stability of the pBtoxis plasmid in conjunction with an upstream DNA–adaptor protein (TubR) and DNAbinding site (tubC). We have determined that TubZ–Bt forms four–stranded filaments in the presence of GTP in vitro, whereas TubZ filaments formed with catalytically compromised protein or non-hydrolysable GTP analogs are predominantly two-stranded. These morphological changes were investigated by high–resolution cryo–electron microscopy of both two– and four–stranded TubZ–Bt filaments, as well as various biochemical approaches, and we propose that TubZ–Bt two–stranded filaments that we observed are an intermediate on pathway to four–stranded filament formation. In addition, we have begun preliminary investigations on the interaction of TubR bind to tubC DNA and the interaction of the TubRC complex with TubZ-Bt filaments.

    A minor focus of this manuscript is work on the eukaryotic tubulins, γ–, δ– and ε–tubulin. This work details preliminary attempts at purification of these tubulin family members in order to begin more detailed investigative work on their molecular function. Also included is a published manuscript of work done with Dr. Luke Rice investigating the conformational state of both αβ– and γ–tubulins using solution–state and crystallographic structural studies.

      Cover page: Structural Characterization of Nucleotide Driven Morphological Changes of TubZ Filaments
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2016)
      Eukaryotic initiation factor 3 (eIF3), an essential multi-protein complex involved in translation initiation, is composed of 12 tightly associated subunits in humans. While the overall structure of eIF3 is known, the mechanism of its assembly and structural consequences of dysregulation of eIF3 subunit expression seen in many cancers is largely unknown. Here we show that subunits in eIF3 assemble into eIF3 in an interdependent manner. Assembly of eIF3 is governed primarily by formation of a helical bundle, composed of helices extending C-terminally from PCI-MPN domains in eight subunits. We propose that, while the minimal subcomplex of human-like eIF3 functional for translation initiation in cells consists of subunits a, b, c, f, g, i, and m, numerous other eIF3 subcomplexes exist under circumstances of subunit over- or underexpression. Thus, eIF3 subcomplexes formed or "released" due to dysregulated subunit expression may be determining factors contributing to eIF3-related cancers.
        Cover page: Assembly of eIF3 Mediated by Mutually Dependent Subunit Insertion
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2014)
        Tubulins are a universally conserved protein superfamily that carry out diverse biological roles by assembling filaments with very different architectures. The underlying basis of this structural diversity is poorly understood. Here, we determine a 7.1 Å cryo-electron microscopy reconstruction of the bacteriophage-encoded PhuZ filament and provide molecular-level insight into its cooperative assembly mechanism. The PhuZ family of tubulins is required to actively center the phage within infected host cells, facilitating efficient phage replication. Our reconstruction and derived model reveal the first example of a three-stranded tubulin filament. We show that the elongated C-terminal tail simultaneously stabilizes both longitudinal and lateral interactions, which in turn define filament architecture. Identified interaction surfaces are conserved within the PhuZ family, and their mutagenesis compromises polymerization in vitro and in vivo. Combining kinetic modeling of PhuZ filament assembly and structural data, we suggest a common filament structure and assembly mechanism for the PhuZ family of tubulins.
          Cover page: The Structure and Assembly Mechanism of a Novel Three-Stranded Tubulin Filament that Centers Phage DNA
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2022)
          Magnetosomes are lipid-bound organelles that direct the biomineralization of magnetic nanoparticles in magnetotactic bacteria. Magnetosome membranes are not uniform in size and can grow in a biomineralization-dependent manner. However, the underlying mechanisms of magnetosome membrane growth regulation remain unclear. Using cryoelectron tomography, we systematically examined mutants with defects at various stages of magnetosome formation to identify factors involved in controlling membrane growth. We found that a conserved serine protease, MamE, plays a key role in magnetosome membrane growth regulation. When the protease activity of MamE is disrupted, magnetosome membrane growth is restricted, which, in turn, limits the size of the magnetite particles. Consistent with this finding, the upstream regulators of MamE protease activity, MamO and MamM, are also required for magnetosome membrane growth. We then used a combination of candidate and comparative proteomics approaches to identify Mms6 and MamD as two MamE substrates. Mms6 does not appear to participate in magnetosome membrane growth. However, in the absence of MamD, magnetosome membranes grow to a larger size than the wild type. Furthermore, when the cleavage of MamD by MamE protease is blocked, magnetosome membrane growth and biomineralization are severely inhibited, phenocopying the MamE protease-inactive mutant. We therefore propose that the growth of magnetosome membranes is controlled by a protease-mediated switch through processing of MamD. Overall, our work shows that, like many eukaryotic systems, bacteria control the growth and size of biominerals by manipulating the physical properties of intracellular organelles.
            Cover page: A protease-mediated switch regulates the growth of magnetosome organelles in Magnetospirillum magneticum
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2012)
            Tubulins are essential for the reproduction of many eukaryotic viruses, but historically, bacteriophage were assumed not to require a cytoskeleton. Here, we identify a tubulin-like protein, PhuZ, from bacteriophage 201φ2-1 and show that it forms filaments in vivo and in vitro. The PhuZ structure has a conserved tubulin fold, with an unusual, extended C terminus that we demonstrate to be critical for polymerization in vitro and in vivo. Longitudinal packing in the crystal lattice mimics packing observed by EM of in-vitro-formed filaments, indicating how interactions between the C terminus and the following monomer drive polymerization. PhuZ forms a filamentous array that is required for positioning phage DNA within the bacterial cell. Correct positioning to the cell center and optimal phage reproduction only occur when the PhuZ filament is dynamic. Thus, we show that PhuZ assembles a spindle-like array that functions analogously to the microtubule-based spindles of eukaryotes.
              Cover page: A Phage Tubulin Assembles Dynamic Filaments by an Atypical Mechanism to Center Viral DNA within the Host Cell
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2019)
              PD-L1 on the surface of tumor cells binds its receptor PD-1 on effector T cells, thereby suppressing their activity. Antibody blockade of PD-L1 can activate an anti-tumor immune response leading to durable remissions in a subset of cancer patients. Here, we describe an alternative mechanism of PD-L1 activity involving its secretion in tumor-derived exosomes. Removal of exosomal PD-L1 inhibits tumor growth, even in models resistant to anti-PD-L1 antibodies. Exosomal PD-L1 from the tumor suppresses T cell activation in the draining lymph node. Systemically introduced exosomal PD-L1 rescues growth of tumors unable to secrete their own. Exposure to exosomal PD-L1-deficient tumor cells suppresses growth of wild-type tumor cells injected at a distant site, simultaneously or months later. Anti-PD-L1 antibodies work additively, not redundantly, with exosomal PD-L1 blockade to suppress tumor growth. Together, these findings show that exosomal PD-L1 represents an unexplored therapeutic target, which could overcome resistance to current antibody approaches.
                Cover page: Suppression of Exosomal PD-L1 Induces Systemic Anti-tumor Immunity and Memory.
                • Article
                • Peer Reviewed
                UC Berkeley Previously Published Works (2023)
                Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-Gs complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.
                  Cover page: Negative allosteric modulation of the glucagon receptor by RAMP2
                  • Article
                  • Peer Reviewed
                  UC Davis Previously Published Works (2022)
                  Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases1,2. A key commitment step is when the ribosomal subunits join at a translation start site on a messenger RNA to form a functional ribosome. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when the universally conserved eukaryotic initiation factors eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we visualized initiation complexes that contained both eIF1A and eIF5B using single-particle cryo-electron microscopy. The resulting structure revealed how eukaryote-specific contacts between the two proteins remodel the initiation complex to orient the initiator aminoacyl-tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during translation initiation in humans.
                    Cover page: eIF5B and eIF1A reorient initiator tRNA to allow ribosomal subunit joining
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