Zhiheng Yu

Integrative structure and functional anatomy of a nuclear pore complex

Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full structural and functional elucidation. Here we determined the structure of the entire 552-protein nuclear pore complex of the yeast Saccharomyces cerevisiae at sub-nanometre precision by satisfying a wide range of data relating to the molecular arrangement of its constituents. The nuclear pore complex incorporates sturdy diagonal columns and connector cables attached to these columns, imbuing the structure with strength and flexibility. These cables also tie together all other elements of the nuclear pore complex, including membrane-interacting regions, outer rings and RNA-processing platforms. Inwardly directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized into distinct functional units. This integrative structure enables us to rationalize the architecture, transport mechanism and evolutionary origins of the nuclear pore complex.

A Near-Atomic Structure of the Dark Apoptosome Provides Insight into Assembly and Activation

In Drosophila, the Apaf-1-related killer (Dark) forms an apoptosome that activates procaspases. To investigate function, we have determined a near-atomic structure of Dark double rings using cryo-electron microscopy. We then built a nearly complete model of the apoptosome that includes 7- and 8-blade β-propellers. We find that the preference for dATP during Dark assembly may be governed by Ser325, which is in close proximity to the 2 carbon of the deoxyribose ring. Interestingly, β-propellers in V-shaped domains of the Dark apoptosome are more widely separated, relative to these features in the Apaf-1 apoptosome. This wider spacing may be responsible for the lack of cytochrome c binding to β-propellers in the Dark apoptosome. Our structure also highlights the roles of two loss-of-function mutations that may block Dark assembly. Finally, the improved model provides a framework to understand apical procaspase activation in the intrinsic cell death pathway.

Correction: Structural basis for the prion-like MAVS filaments in antiviral innate immunity

Mitochondrial antiviral signaling (MAVS) protein forms prion-like aggregates mediated by the N-terminal caspase activation and recruitment domain (CARD) and activates antiviral signaling cascades. Purified MAVS CARD from culture cells self-assembles into filaments. Previously, we reported a low-resolution cryoEM structure of MAVS CARD filament, which exhibits a C3 symmetry with a rotation of −53.6° and an axial rise of 16.8 A for every unit in the filament (Xu et al., 2014). Recently, a cryoEM reconstruction of MAVS CARD filaments at 3.6 A resolution was reported with a C1 helical symmetry of a rotation of −101.1° and an axial rise of 5.1 A per subunit (Wu et al., 2014). The differences in these two models were carefully analyzed recently (Egelman, 2014), which suggested that the helical ambiguity in helical reconstruction was not fully resolved in our previous analysis (Xu et al., 2014). We recently collected a new dataset at higher resolutions. Using a newly developed method for analysis of helical filaments (Clemens et al., 2015), we obtained a 4.2 A resolution reconstruction of MAVS CARD filaments purified from mammalian cells under native conditions. The new model shows that the MAVS CARD filament exhibits a C1 helical symmetry in agreement with Wu et al. (2014).

Structural Basis for the Prion-Like Mavs Filaments in Antiviral Innate Immunity

Mitochondrial anti-viral signaling (MAVS) protein is a critical adaptor required for innate immune responses against RNA viruses. In virus-infected cells MAVS forms prion-like aggregates to activate antiviral signaling cascades, but the structural mechanism underlying such aggregation is unknown. Here we report cryo-electron microscopic structures of the helical filaments formed by both the N- terminal caspase activation and recruitment domain of MAVS and a truncated MAVS lacking its C-terminal transmembrane domain. Both structures display a left-handed three-stranded helical filament, revealing specific interfaces between individual subunits that are dictated by electrostatic interactions between neighboring strands and conserved hydrophobic interactions within each strand. Point mutations at multiple locations of these two interfaces impaired filament formation and antiviral signaling. Super-resolution imaging of virus-infected cells revealed the spatial features of rod-shaped MAVS clusters on mitochondria. These results elucidate the structural mechanism of MAVS polymerization, and explain how an α-helical domain uses distinct chemical interactions to form self-perpetuating filaments.