Qiu-Xing Jiang

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.

Secretory granule protein chromogranin B (CHGB) forms an anion channel in membranes

The CHGB subfamily of secretory granule proteins forms a new family of anion-selective channels by interacting with membranes via two amphipathic α-helices. The channel exhibits higher anion selectivity, larger conductance, higher DIDS-binding affinity, and higher Cl− sensitivity than other known anion channels. Regulated secretion is an intracellular pathway that is highly conserved from protists to humans. Granin family proteins were proposed to participate in the biogenesis, maturation and release of secretory granules in this pathway. However, the exact molecular mechanisms underlying the intracellular functions of the granin family proteins remain unclear. Here, we show that chromogranin B (CHGB), a secretory granule protein, inserts itself into membrane and forms a chloride-conducting channel. CHGB interacts strongly with phospholipid membranes through two amphipathic α helices. At a high local concentration, CHGB insertion in membrane causes significant bilayer remodeling, producing protein-coated nanoparticles and nanotubules. Fast kinetics and high cooperativity for anion efflux from CHGB vesicles suggest that CHGB tetramerizes to form a functional channel with a single-channel conductance of ∼125 pS (150/150 mM Cl−). The CHGB channel is sensitive to an anion channel blocker and exhibits higher anion selectivity than the other six known families of Cl− channels. Our data suggest that the CHGB subfamily of granin proteins forms a new family of organelle chloride channels.

Electron Crystallography Reveals a Possible Motion Mechanism for the Voltage Sensor Domains in Membranes

Structural and functional studies of voltage-gated ion channels have showed that the voltage sensor domains (VSDs) are allosteric nanomachines that take two distinct gating conformations and require lipid molecules for their normal function. The four-helix bundle conformer of the VSDs seen in both Kv1.2 and NavAb structures in detergent micelles is currently thought to reflect the “UP” (activated or open) conformation of the VSDs. It remains unclear how a VSD physically switches between different conformational states in a voltage-gated channel. To provide more insights on the conformational switch of the VSDs in membranes, we have been working on 2D crystals of the prototype KvAP channels in different lipid membranes. We identified conditions that retain the KvAP VSDs in specific conformations, and have optimized one crystal form to better than 6 A. Our electron crystallographic map of the channel in the inactivated state revealed a VSD conformation that is significantly different from the four-helix bundle. It demonstrated that the four helices in a VSD are capable of moving laterally apart and becoming stretched around the pore domain. Our cysteine-based crosslinking experiments at various sites support that in lipid membranes, the VSDs can become splayed open so that single cysteine residues introduced in the first or fourth helices can form disulfides with themselves, and specific sites from neighboring VSDs in one tetrameric channel can get into close proximity with each other. Our results suggest that there are distinct “UP” conformations for a VSD in membranes and that the switch between splayed-open and four-helix bundle conformations in a VSD could be an important mechanism for its gating movement.

Phospholipids as a Structural and Functional Determinant for Voltage Sensors in a Kv Channel

Recent studies proposed that phosphodiester groups in a phospholipid bilayer help stabilize a voltage sensor in its activated conformation. However, the nature of such channel-lipid interaction is unclear. We are studying the structure and function of the KvAP channel in lipid bilayers. We obtain the structural information of the channel in membranes by electron crystallographic study of two-dimensional crystals, and utilize both biochemical assays and electrical recordings to determine the conformational states of the voltage sensors and the pore domain in reconstituted channels. Our electron crystallographic studies start to reveal that the KvAP voltage sensors in a phospholipid membrane can take an alternative conformation, which is different from the expected four-helix bundle structure observed in the X-ray crystallographic studies of Kv channel proteins in detergents or mixed detergent/lipid micelles. The functional implications of such a new conformation of the voltage sensor are being investigated. Our functional studies converge to the conclusion that annular phospholipids around the channel are required for its voltage sensors to switch from the deactivated to the activated state. Our data suggest that a phospholipid bilayer may be an essential factor for the structure and function of a voltage sensor.