Chemotactic responses in bacteria require large, highly ordered arrays of sensory proteins to mediate the signal transduction that ultimately controls cell motility. A mechanistic understanding of the molecular events underlying signaling, however, has been hampered by the lack of a high-resolution structural description of the extended array. Here, we report a novel reconstitution of the array, involving the receptor signaling domain, histidine kinase CheA, and adaptor protein CheW, as well as a density map of the core-signaling unit at 11.3 Å resolution, obtained by cryo-electron tomography and sub-tomogram averaging. Extracting key structural constraints from our density map, we computationally construct and refine an atomic model of the core array structure, exposing novel interfaces between the component proteins. Using all-atom molecular dynamics simulations, we further reveal a distinctive conformational change in CheA. Mutagenesis and chemical cross-linking experiments confirm the importance of the conformational dynamics of CheA for chemotactic function.

Summary The myxovirus resistance (Mx) proteins are interferon-induced dynamin GTPases that can inhibit a variety of viruses. Recently, MxB, but not MxA, was shown to restrict HIV-1 by an unknown mechanism that likely occurs in close proximity to the host cell nucleus and involves the viral capsid. Here, we present the crystal structure of MxB and reveal determinants involved in HIV-1 restriction. MxB adopts an extended anti-parallel dimer and dimerization, but not higher-ordered oligomerization, is critical for restriction. Although MxB is structurally similar to MxA, the orientation of individual domains differs between MxA and MxB and their antiviral functions rely on separate determinants, indicating distinct mechanisms for virus inhibition. Additionally, MxB directly binds the HIV-1 capsid and this interaction depends on dimerization and the N-terminus of MxB as well as the assembled capsid lattice. These insights establish a framework for understanding the mechanism by which MxB restricts HIV-1.

Background: Drp1 oligomerization and activity is critical for mitochondrial fission. Results: GTP hydrolysis is required for Drp1 constriction of lipid bilayers. The variable domain of Drp1 regulates self-assembly and is not required for constriction of lipid bilayers. Conclusion:The core machinery of Drp1 is sufficient to mediate lipid assembly, constriction, and disassembly. Significance: Characterization of the mechanoenzymatic properties of Drp1 advances our understanding of mitochondrial fission.

CryoEM structure of MxB tubes at 4.6 Å resolution reveals novel interfaces responsible for assembly and anti–HIV-1 activity.

Transmembrane (TM) signaling involves the interaction of membrane-spanning proteins with ligands or proteins on both sides of the membrane. The well-characterized maltose transporter (MalFGK 2 ) from E. coli, an ATP binding cassette (ABC) transporter, is one such system. The interaction of a periplasmic maltose binding protein (MBP) with the TM subunits (MalF-MalG) of the transporter stimulates the ATPase activity of the MalK dimer at the cytoplasmic surface of the membrane.1 Biophysical studies of purified membrane proteins are traditionally performed either in detergent micelles or in proteoliposome vesicles.2 However, each has a weakness. The insertion of proteins into liposomes generates heterogeneity in orientation and in accessibility to ligands, while detergent micelles can be poor membrane mimics. Work by Grote et al.3 using electron paramagnetic resonance (EPR) spectroscopy illustrates how 50% of the population of MalFGK 2 can fail to respond to the addition of nucleotide to proteoliposomes, presumably because the nucleotide-binding sites face the lumen. MalFGK 2 displays an MBP-independent ATPase activity in detergent that is not characteristic of the reconstituted transporter.4 We demonstrate here that nanodiscs5 , 6 provide an ideal solution to these problems and are nicely suited for EPR spectroscopy in the investigation of the structural dynamics of multi-spanning TM proteins.A nanodisc consists of two membrane scaffold proteins (MSPs), modeled after the serum apoprotein A-1, encircling a patch of phospholipid bilayer. Plasmids are commercially available encoding MSPs of different sizes, ranging from 9.8 to 17.0 nm in diameter.7 Incorporation of the TM region of a membrane protein into the lipid patch renders the protein soluble in aqueous solution in the absence of detergent and exposes both hydrophilic surfaces to solution. The MalFGK 2 transporter was reconstituted into nanodiscs following the procedures outlined (supplementary information) to determine whether the characteristic ATPase activity seen in proteoliposomes is faithfully recapitulated in nanodiscs.Optimization of nanodisc assembly was achieved by varying the molar ratios of MSP, transporter and lipids. Soybean phospholipids, previously found to be suitable for reconstitution of MalFGK 2 , 8 was solubilized with cholate, a detergent commonly used in the nanodiscs literature.7 N-dodecyl-β-D-maltoside (DDM), a gentle nonionic detergent routinely used for stabilization of membrane proteins9 worked equally well (data not shown). Pure lipids and E. coli phospholipids have also been used in nanodiscs.7 , 10 Initially, nanodiscs were prepared using a 120:1 molar ratio of lipids/MSP with MSP(monomer)/transporter ratios varying from 1:1 to 20:1. A high MSP/transporter ratio increases the likelihood of incorporating just one transporter per nanodisc,10 , 11 while a lower ratio reduces the amount of empty nanodiscs and economizes on the use of MSP. Nanodiscs were taken for assay directly following detergent removal by biobeads (crude NIH-PA Aut...