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Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2024)

As we seek to elucidate the functions and mechanisms of biological constructs, there is a growing need to learn about their structures in their native contexts as members of their molecular societies. Conventional methods of structural biology entail purification of recombinant proteins, which isolates them from their interactome and spatial localization. Thus, minimizing sample manipulation is vital for discovering biological interactions. Unique among the structural biology methods, cryogenic electron microscopy (cryoEM) is able to generate data from impure, heterogeneous samples. By combining old-fashioned endogenous methods with recent advancements in the throughput of cryoEM data collection and computation, we can perform purification in silico, allowing for high resolution structural determination of proteins while retaining their native binding partners. We apply this endogenous cryoEM approach to determine the structures of two critical metabolic complexes, the icosahedral pyruvate dehydrogenase complex (PDC) and the cubic 2-oxoglutarate dehydrogenase complex (OGDC), from mitochondrial lysates of bovine kidneys. By minimizing disruption of the sample, we identify heterogeneous arrangements of E1 and E3 peripheral subunits bound to their central E2 cores. Additionally, the substrate-shuttling lipoyl moiety of E2 is found within the E2 active site. Direct comparison of PDC and OGDC from the same organism allows us to identify critical interaction interfaces for the prevention of heterologous binding and determination of geometry despite a near-identical monomeric E2 fold. Some systems do not have the luxury of containing multiple copies for averaging by single particle cryoEM. Instead, cryogenic electron tomography (cryoET) enables imaging of unique subjects within their fully intact environment. Here, we report structures and observations of intact cancer-causing herpesviruses, Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV), and their pleiomorphic tegument and envelope using cryoET. Within their teguments, we observe strand-like features that suggest a means of viral assembly. We also show that herpesvirus fusogen proteins assemble into triplet clusters to promote host membrane fusion. Our results provide new insights into assembly of metabolic complexes and herpesviruses. Together, they highlight wide applicability of cryoEM and cryoET for understanding large complexes in context of their native systems.

    Cover page: Structures from cells and viruses through endogenous cryoEM methods
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2021)
    Aβ42 aggregation plays a central role in the pathogenesis of Alzheimer's disease. In addition to the insoluble fibrils that comprise the amyloid plaques, Aβ42 also forms soluble aggregates collectively called oligomers, which are more toxic and pathogenic than fibrils. Understanding the structure and dynamics of Aβ42 oligomers is critical for developing effective therapeutic interventions against these oligomers. Here we studied the structural dynamics of Aβ42 globulomers, a type of Aβ42 oligomers prepared in the presence of sodium dodecyl sulfate, using site-directed spin labeling. Spin labels were introduced, one at a time, at all 42 residue positions of Aβ42 sequence. Electron paramagnetic resonance spectra of spin-labeled samples reveal four structural segments based on site-dependent spin label mobility pattern. Segment-1 consists of residues 1-6, which have the highest mobility that is consistent with complete disorder. Segment-3 is the most immobilized region, including residues 31-34. Segment-2 and -4 have intermediate mobility and are composed of residues 7-30 and 35-42, respectively. Considering the inverse relationship between protein dynamics and stability, our results suggest that residues 31-34 are the most stable segment in Aβ42 oligomers. At the same time, the EPR spectral lineshape suggests that Aβ42 globulomers lack a well-packed structural core akin to that of globular proteins.
      Cover page: Segmental structural dynamics in Aβ42 globulomers.
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2022)
      The α-keto acid dehydrogenase complex family catalyzes the essential oxidative decarboxylation of α-keto acids to yield acyl-CoA and NADH. Despite performing the same overarching reaction, members of the family have different component structures and structural organization between each other and across phylogenetic species. While native structures of α-keto acid dehydrogenase complexes from bacteria and fungi became available recently, the atomic structure and organization of their mammalian counterparts in native states remain unknown. Here, we report the cryo-electron microscopy structures of the endogenous cubic 2-oxoglutarate dehydrogenase complex (OGDC) and icosahedral pyruvate dehydrogenase complex (PDC) cores from bovine kidney determined at resolutions of 3.5 Å and 3.8 Å, respectively. The structures of multiple proteins were reconstructed from a single lysate sample, allowing direct structural comparison without the concerns of differences arising from sample preparation and structure determination. Although native and recombinant E2 core scaffold structures are similar, the native structures are decorated with their peripheral E1 and E3 subunits. Asymmetric sub-particle reconstructions support heterogeneity in the arrangements of these peripheral subunits. In addition, despite sharing a similar monomeric fold, OGDC and PDC E2 cores have distinct interdomain and intertrimer interactions, which suggests a means of modulating self-assembly to mitigate heterologous binding between mismatched E2 species. The lipoyl moiety lies near a mobile gatekeeper within the interdomain active site of OGDC E2 and PDC E2. Analysis of the twofold related intertrimer interface identified secondary structural differences and chemical interactions between icosahedral and cubic geometries of the core. Taken together, our study provides a direct structural comparison of OGDC and PDC from the same source and offers new insights into determinants of interdomain interactions and of architecture diversity among α-keto acid dehydrogenase complexes.
        Cover page: Structures and comparison of endogenous 2-oxoglutarate and pyruvate dehydrogenase complexes from bovine kidney
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2022)
        7SK non-coding RNA (7SK) negatively regulates RNA polymerase II (RNA Pol II) elongation by inhibiting positive transcription elongation factor b (P-TEFb), and its ribonucleoprotein complex (RNP) is hijacked by HIV-1 for viral transcription and replication. Methylphosphate capping enzyme (MePCE) and La-related protein 7 (Larp7) constitutively associate with 7SK to form a core RNP, while P-TEFb and other proteins dynamically assemble to form different complexes. Here, we present the cryo-EM structures of 7SK core RNP formed with two 7SK conformations, circular and linear, and uncover a common RNA-dependent MePCE-Larp7 complex. Together with NMR, biochemical, and cellular data, these structures reveal the mechanism of MePCE catalytic inactivation in the core RNP, unexpected interactions between Larp7 and RNA that facilitate a role as an RNP chaperone, and that MePCE-7SK-Larp7 core RNP serves as a scaffold for switching between different 7SK conformations essential for RNP assembly and regulation of P-TEFb sequestration and release.
          Cover page: Structural basis of RNA conformational switching in the transcriptional regulator 7SK RNP
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2023)
          Infection by Kaposi sarcoma-associated herpesvirus (KSHV) can cause severe consequences, such as cancers and lymphoproliferative diseases. Whole inactivated viruses (WIV) with chemically destroyed genetic materials have been used as antigens in several licensed vaccines. During KSHV productive replication, virus-like vesicles (VLVs) that lack capsids and viral genomes are generated along with virions. Here, we investigated the immunogenicity of KSHV VLVs produced from a viral mutant that was defective in capsid formation and DNA packaging. Mice immunized with adjuvanted VLVs generated KSHV-specific T cell and antibody responses. Neutralization of KSHV infection by the VLV immune serum was low but was markedly enhanced in the presence of the complement system. Complement-enhanced neutralization and complement deposition on KSHV-infected cells was dependent on antibodies targeting viral open reading frame 4 (ORF4). However, limited complement-mediated enhancement was detected in the sera of a small cohort of KSHV-infected humans which contained few neutralizing antibodies. Therefore, vaccination that induces antibody effector functions can potentially improve infection-induced humoral immunity. Overall, our study highlights a potential benefit of engaging complement-mediated antibody functions in future KSHV vaccine development. IMPORTANCE KSHV is a virus that can lead to cancer after infection. A vaccine that prevents KSHV infection or transmission would be helpful in preventing the development of these cancers. We investigated KSHV VLV as an immunogen for vaccination. We determined that antibodies targeting the viral protein ORF4 induced by VLV immunization could engage the complement system and neutralize viral infection. However, ORF4-specific antibodies were seldom detected in the sera of KSHV-infected humans. Moreover, these human sera did not potently trigger complement-mediated neutralization, indicating an improvement that immunization can confer. Our study suggests a new antibody-mediated mechanism to control KSHV infection and underscores the benefit of activating the complement system in a future KSHV vaccine.
            Cover page: Immunization of Mice with Virus-Like Vesicles of Kaposi Sarcoma-Associated Herpesvirus Reveals a Role for Antibodies Targeting ORF4 in Activating Complement-Mediated Neutralization
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