Selected Publications:
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Cholesterol regulates plasma membrane bending by prominin-family proteins
Tristan A. Bell^, Bridget E. Luce, Pusparanee Hakim, Hiba Dardari, Virly Y. Ananda, Tran H. Nguyen, Arezu Monshizadeh, Luke H. Chao^ bioRxiv 2023.11.08.566258v2; doi: https://doi.org/10.1101/2023.11.08.566258 ^co-corresponding Prominin-1 (Prom1) is a pentaspan membrane protein that associates with curved regions of the plasma membrane. Prom1 localizes to cholesterol-rich domains and requires membrane cholesterol to support membrane remodeling. Membrane bending activity is particularly evident in photoreceptors, where Prom1 mutations cause loss of outer segment disk homeostasis leading to cone-rod retinal dystrophy (CCRD). However, the mechanistic link between prominin-dependent cholesterol binding, membrane remodeling, and retinal disease remains unclear. Here, we characterize the membrane bending function and specific cholesterol binding activity of Prom1 and its proposed homolog Tweety homology 1 (Ttyh1) in extracellular vesicles (EVs). Prom1 and Ttyh1 induce formation of EVs in cultured mammalian cells that are biophysically similar. Though both proteins bend membranes and form EVs at the plasma membrane, Ttyh1 lacks a stable interaction with cholesterol that is present in Prom1. Correspondingly, Ttyh1 forms EVs that are more deformed than those produced by Prom1. An evolutionarily conserved and retinal disease-associated Prom1 residue (Trp-795) is necessary for cholesterol binding, EV membrane deformation, and efficient trafficking to the plasma membrane. Removal of N-glycan moieties from Prom1 biases the enzyme toward a cholesterol-bound state. We propose that Prom1 and Ttyh1 are both members of a single prominin family of membrane bending proteins, that Ttyh1 is a constitutively active member of this family, and that Prom1 is regulated by cholesterol binding and N-glycosylation. These findings shed light on mechanisms of prominin family function in disease and help unify models of prominin function across diverse cell types. |
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Helical reconstruction of VP39 reveals principles for baculovirus nucleocapsid assembly
Friederike M. C. Benning, Simon Jenni, Coby Y. Garcia, Tran H. Nguyen, Xuewu Zhang, Luke H. Chao bioRxiv 2023.06.15.545104 PDF doi: https://doi.org/10.1101/2023.06.15.545104 Baculoviruses are insect-infecting pathogens with wide applications as biological pesticides, in vitro protein production vehicles and gene therapy tools. Its cylindrical nucleocapsid, which encapsulates and protects the circular double-stranded viral DNA encoding proteins for viral replication and entry, is formed by the highly conserved major capsid protein VP39. The mechanism for VP39 assembly remains unknown. We determined a 3.2 Å electron cryomicroscopy helical reconstruction of an infectious nucleocapsid of Autographa californica multiple nucleopolyhedrovirus, revealing how dimers of VP39 assemble into a 14-stranded helical tube. We show that VP39 comprises a unique protein fold conserved across baculoviruses, which includes a Zinc finger domain and a stabilizing intra-dimer sling. Analysis of sample polymorphism revealed tube flattening could account for different helical geometries. This VP39 reconstruction reveals general principles for baculoviral nucleocapsid assembly. |
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In situ architecture of Opa1-dependent mitochondrial cristae remodeling
Michelle Y. Fry*, Paula P. Navarro*, Xingping Qin, Zintes Inde, Virly Y. Ananda, Camila Makhlouta Lugo, Pusparanee Hakim, Bridget E. Luce, Yifan Ge, Julie L. McDonald, Ilzat Ali, Leillani L. Ha, Benjamin P. Kleinstiver, David C. Chan, Kristopher Sarosiek, Luke H. Chao bioRxiv 2023.01.16.524176 PDF doi: https://www.biorxiv.org/content/10.1101/2023.01.16.524176v1 *equal contribution Cristae membrane state plays a central role in regulating mitochondrial function and cellular metabolism. The protein Optic atrophy 1 (Opa1) is an important crista remodeler that exists as two forms in the mitochondrion, a membrane-anchored long form (l-Opa1) and a processed short form (s-Opa1). The mechanisms for how Opa1 influences cristae shape have remained unclear due to the lack of native 3D views of cristae morphology. We perform in situ cryo-electron tomography of cryo-focused ion beam milled mouse embryonic fibroblasts to understand how each form of Opa1 influences cristae architecture. In our tomograms, we observe elongated mitochondria with a stacking phenotype, and an absence of tubular cristae, when only l-Opa1 is present. In contrast, when mitochondria contain mainly s-Opa1, we observe irregular cristae packing, an increase in globular cristae, and decreased matrix condensation. Notably, we find the absence of l-Opa1 results in mitochondria with wider cristae junctions. BH3 profiling reveals that absence of l-Opa1 reduces cytochrome c release in response to pro-apoptotic stimuli. We discuss the implications Opa1-dependent cristae morphologies in cell death initiation. |
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Cell wall synthesis and remodeling dynamics determine bacterial division site architecture and cell shape
Paula P. Navarro*, Andrea Vettiger*, Virly Y Ananda, Paula Montero Llopis, Christoph Allolio, Thomas G Bernhardt^, Luke H Chao^ Nature Microbiology, (2022). https://doi.org/10.1038/s41564-022-01210-z PDF *equal contribution ^co-corresponding The bacterial division apparatus catalyses the synthesis and remodelling of septal peptidoglycan (sPG) to build the cell wall layer that fortifies the daughter cell poles. Understanding of this essential process has been limited by the lack of native three-dimensional views of developing septa. Here, we apply state-of-the-art cryogenic electron tomography (cryo-ET) and fluorescence microscopy to visualize the division site architecture and sPG biogenesis dynamics of the Gram-negative bacterium Escherichia coli. We identify a wedge-like sPG structure that fortifies the ingrowing septum. Experiments with strains defective in sPG biogenesis revealed that the septal architecture and mode of division can be modified to more closely resemble that of other Gram-negative (Caulobacter crescentus) or Gram-positive (Staphylococcus aureus) bacteria, suggesting that a conserved mechanism underlies the formation of different septal morphologies. Finally, analysis of mutants impaired in amidase activation (ΔenvC ΔnlpD) showed that cell wall remodelling affects the placement and stability of the cytokinetic ring. Taken together, our results support a model in which competition between the cell elongation and division machineries determines the shape of cell constrictions and the poles they form. They also highlight how the activity of the division system can be modulated to help generate the diverse array of shapes observed in the bacterial domain. |
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Absence of cardiolipin from the mitochondrial inner membrane outer leaflet restricts Opa1-mediated fusion
Yifan Ge, Sivakumar Boopathy, Tran H Nguyen, Camila Makhlouta Lugo, Luke H Chao Front. Mol. Biosci., 22 December 2021 doi: https://doi.org/10.3389/fmolb.2021.769135 PMID: 35004847 PDF Cardiolipin is a tetra-acylated di-phosphatidylglycerol lipid enriched in the matrix-facing (inner) leaflet of the mitochondrial inner membrane. Cardiolipin plays an important role in regulating mitochondria function and dynamics. Yet, the mechanisms connecting cardiolipin distribution and mitochondrial protein function remain indirect. In our previous work, we established an in vitro system reconstituting mitochondrial inner membrane fusion mediated by Opa1. We found that the long form of Opa1 (l-Opa1) works together with the proteolytically processed short form (s-Opa1) to mediate fast and efficient membrane fusion. Here, we extend our reconstitution system to generate supported lipid bilayers with asymmetric cardiolipin distribution. Using this system, we find the presence of cardiolipin on the inter-membrane space-facing (outer) leaflet is important for membrane tethering and fusion. We discuss how the presence of cardiolipin in this leaflet may influence protein and membrane properties, and future applications for this approach. |
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A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics
Yifan Ge, Sivakumar Boopathy , Adam Smith, Luke H Chao J Vis Exp. 2020 Sep 2;(163). doi: 10.3791/61620. PMID: 32955498 PDF Video Mitochondrial dynamics is essential for the organelle's diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems. |
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Two forms of Opa1 cooperate to complete fusion of the mitochondrial inner-membrane
Yifan Ge, Xiaojun Shi, Sivakumar Boopathy, Julie McDonald, Adam W Smith, Luke H Chao eLife 2020; 9:e50973 PMID: 31922487 PDF Mitochondrial membrane dynamics is a cellular rheostat that relates metabolic function and organelle morphology. Using an in vitro reconstitution system, we describe a mechanism for how mitochondrial inner-membrane fusion is regulated by the ratio of two forms of Opa1. We found that the long-form of Opa1 (l-Opa1) is sufficient for membrane docking, hemifusion and low levels of content release. However, stoichiometric levels of the processed, short form of Opa1 (s-Opa1) work together with l-Opa1 to mediate efficient and fast membrane pore opening. Additionally, we found that excess levels of s-Opa1 inhibit fusion activity, as seen under conditions of altered proteostasis. These observations describe a mechanism for gating membrane fusion. |
Highlights from PhD and Postdoctoral work:
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How small-molecule inhibitors of dengue-virus infection interfere with viral membrane fusion
L.H. Chao, J. Jang, A. Johnson, A. Nguyen, N.S. Gray, P.L. Yang, S.C. Harrison eLife 2018;7:e36461 PMID: 29999491 PDF Dengue virus (DV) is a compact, icosahedrally symmetric, enveloped particle, covered by 90 dimers of envelope protein (E), which mediates viral attachment and membrane fusion. Fusion requires a dimer-to-trimer transition and membrane engagement of hydrophobic 'fusion loops'. We previously characterized the steps in membrane fusion for the related West Nile virus (WNV), using recombinant, WNV virus-like particles (VLPs) for single-particle experiments (Chao et al., 2014). Trimerization and membrane engagement are rate-limiting; fusion requires at least two adjacent trimers; availability of competent monomers within the contact zone between virus and target membrane creates a trimerization bottleneck. We now report an extension of that work to dengue VLPs, from all four serotypes, finding an essentially similar mechanism. Small-molecule inhibitors of dengue virus infection that target E block its fusion-inducing conformational change. We show that ~12-14 bound molecules per particle (~20-25% occupancy) completely prevent fusion, consistent with the proposed mechanism. |
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Sequential conformational rearrangements in flavivirus membrane fusion
L.H. Chao, D.E. Klein, A.G. Schmidt, J.M. Peña, S.C. Harrison eLife 2014;3:e04389 PMID: 25479384 PDF The West Nile Virus (WNV) envelope protein, E, promotes membrane fusion during viral cell entry by undergoing a low-pH triggered conformational reorganization. We have examined the mechanism of WNV fusion and sought evidence for potential intermediates during the conformational transition by following hemifusion of WNV virus-like particles (VLPs) in a single particle format. We have introduced specific mutations into E, to relate their influence on fusion kinetics to structural features of the protein. At the level of individual E subunits, trimer formation and membrane engagement of the threefold clustered fusion loops are rate-limiting. Hemifusion requires at least two adjacent trimers. Simulation of the kinetics indicates that availability of competent monomers within the contact zone between virus and target membrane makes trimerization a bottleneck in hemifusion. We discuss the implications of the model we have derived for mechanisms of membrane fusion in other contexts. |
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A mechanism for tunable autoinhibition in the structure of a human Ca2+/calmodulin-dependent kinase II holoenzyme
L.H. Chao, M.M. Stratton, I-H. Lee, O.S. Rosenberg, J. Levitz, D.J. Mandell, T. Kortemme, J.T. Groves, H. Schulman, J. Kuriyan Cell, 2011 Sep 146(5): 732-45 PMC3184253, PDB: 3SOA, Cell Paperflick video abstract PDF Calcium/calmodulin-dependent kinase II (CaMKII) forms a highly conserved dodecameric assembly that is sensitive to the frequency of calcium pulse trains. Neither the structure of the dodecameric assembly nor how it regulates CaMKII are known. We present the crystal structure of an autoinhibited full-length human CaMKII holoenzyme, revealing an unexpected compact arrangement of kinase domains docked against a central hub, with the calmodulin-binding sites completely inaccessible. We show that this compact docking is important for the autoinhibition of the kinase domains and for setting the calcium response of the holoenzyme. Comparison of CaMKII isoforms, which differ in the length of the linker between the kinase domain and the hub, demonstrates that these interactions can be strengthened or weakened by changes in linker length. This equilibrium between autoinhibited states provides a simple mechanism for tuning the calcium response without changes in either the hub or the kinase domains. |
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Inter-subunit capture of regulatory segments is a component of cooperative CaMKII activation
L.H. Chao*, P. Pellicena*, S. Deindl, L.A. Barclay, H. Schulman, J. Kuriyan *denotes equal contribution. Nature Structural and Molecular Biology, 2010 Mar 17(3): 264-72 PMC2855215 PDB: 3KK8 3KK9 3KL8 PDF The dodecameric holoenzyme of calcium-calmodulin-dependent protein kinase II (CaMKII) responds to high-frequency Ca(2+) pulses to become Ca(2+) independent. A simple coincidence-detector model for Ca(2+)-frequency dependency assumes noncooperative activation of kinase domains. We show that activation of CaMKII by Ca(2+)-calmodulin is cooperative, with a Hill coefficient of approximately 3.0, implying sequential kinase-domain activation beyond dimeric units. We present data for a model in which cooperative activation includes the intersubunit 'capture' of regulatory segments. Such a capture interaction is seen in a crystal structure that shows extensive contacts between the regulatory segment of one kinase and the catalytic domain of another. These interactions are mimicked by a natural inhibitor of CaMKII. Our results show that a simple coincidence-detection model cannot be operative and point to the importance of kinetic dissection of the frequency-response mechanism in future experiments. |