Proteins containing C2 domains are the sensors for Ca 2+ and PI (4,5)P 2 in a myriad of secretory pathways. Here, the use of a freemounting system has enabled us to capture an intermediate state of Ca 2+ binding to the C2A domain of rabphilin 3A that suggests a different mechanism of ion interaction. We have also determined the structure of this domain in complex with PI(4,5)P 2 and IP 3 at resolutions of 1.75 and 1.9 Å, respectively, unveiling that the polybasic cluster formed by strands β3-β4 is involved in the interaction with the phosphoinositides. A comparative study demonstrates that the C2A domain is highly specific for PI(4,5)P 2 /PI (3,4,5)P 3 , whereas the C2B domain cannot discriminate among any of the diphosphorylated forms. Structural comparisons between C2A domains of rabphilin 3A and synaptotagmin 1 indicated the presence of a key glutamic residue in the polybasic cluster of synaptotagmin 1 that abolishes the interaction with PI (4,5)P 2 . Together, these results provide a structural explanation for the ability of different C2 domains to pull plasma and vesicle membranes close together in a Ca 2+ -dependent manner and reveal how this family of proteins can use subtle structural changes to modulate their sensitivity and specificity to various cellular signals.PIP2 | calcium | vesicle fusion C 2 modules are most commonly found in enzymes involved in lipid modifications and signal transduction and in proteins involved in membrane trafficking. They consist of 130 residues and share a common fold composed of two four-stranded β-sheets arranged in a compact β-sandwich connected by surface loops and helices (1-4). Many of these C2 domains have been demonstrated to function in a Ca 2+ -dependent membrane-binding manner and hence act as cellular Ca 2+ sensors. Calcium ions bind in a cupshaped invagination formed by three loops at one tip of the β-sandwich where the coordination spheres for the Ca 2+ ions are incomplete (5-7). This incomplete coordination sphere can be occupied by neutral and anionic (7-9) phospholipids, enabling the C2 domain to dock at the membrane.Previous work in our laboratory has shed light on the 3D structure of the C2 domain of PKCα in complex with both PS and PI(4,5)P 2 simultaneously (10). This revealed an additional lipidbinding site located in the polybasic region formed by β3-β4 strands that preferentially binds to PI(4,5)P 2 (11-15). This site is also conserved in a wide variety of C2 domains of topology I, for example synaptotagmins, rabphilin 3A, DOC2, and PI3KC2α (10,(16)(17)(18)(19). Given the importance of PI(4,5)P 2 for bringing the vesicle and plasma membranes together before exocytosis to ensure rapid and efficient fusion upon calcium influx (20-23), it is crucial to understand the molecular mechanisms beneath this event.Many studies have reported different and contradictory results about the membrane binding properties of C2A and C2B domains of synaptotagmin 1 and rabphilin 3A providing an unclear picture about how Ca 2+ and PI(4,5)P 2 combine to orchestrate the vesi...

Vaults are the largest ribonucleoprotein particles found in eukaryotic cells, with an unclear cellular function and promising applications as vehicles for drug delivery. In this article, we examine the local stiffness of individual vaults and probe their structural stability with atomic force microscopy under physiological conditions. Our data show that the barrel, the central part of the vault, governs both the stiffness and mechanical strength of these particles. In addition, we induce single-protein fractures in the barrel shell and monitor their temporal evolution. Our high-resolution atomic force microscopy topographies show that these fractures occur along the contacts between two major vault proteins and disappear over time. This unprecedented systematic self-healing mechanism, which enables these particles to reversibly adapt to certain geometric constraints, might help vaults safely pass through the nuclear pore complex and potentiate their role as self-reparable nanocontainers.

Recent studies reveal that the mechanical properties of virus particles may have been shaped by evolution to facilitate virus survival. Manipulation of the mechanical behavior of virus capsids is leading to a better understanding of viral infection, and to the development of virus-based nanoparticles with improved mechanical properties for nanotechnological applications. In the minute virus of mice (MVM), deleterious mutations around capsid pores involved in infection-related translocation events invariably increased local mechanical stiffness and interfered with pore-associated dynamics. To provide atomic-resolution insights into biologically relevant changes in virus capsid mechanics, we have determined by X-ray crystallography the structural effects of deleterious, mechanically stiffening mutations around the capsid pores. Data show that the cavity-creating N170A mutation at the pore wall does not induce any dramatic structural change around the pores, but instead generates subtle rearrangements that propagate throughout the capsid, resulting in a more compact, less flexible structure. Analysis of the spacefilling L172W mutation revealed the same relationship between increased stiffness and compacted capsid structure. Implications for understanding connections between virus mechanics, structure, dynamics and infectivity, and for engineering modified virus-based nanoparticles, are discussed.