Two forms of Opa1 cooperate to complete fusion of the mitochondrial inner-membrane
bioRxiv 10.1101/739078v2 18 August 2019
Mitochondrial membrane dynamics is a cellular rheostat that relates organelle morphology and metabolic function. 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 in conditions of abnormal cellular proteostasis. These observations describe a mechanism for gating membrane fusion.
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 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.
Sequential conformational rearrangements in flavivirus membrane fusion
L.H. Chao, D.E. Klein, A.G. Schmidt, J.M. Peña, S.C. Harrison
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.
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.
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.