GTPase is a key mediator of cell-autonomous innate immunityHis-tagged full-length human MxA (Fig. 1a) was recombinantly expressed in bacteria and purified to homogeneity (Methods, Supp. Fig. 1). In crystallization trials, small needle-shaped protein crystals were obtained which represented proteolytic cleavage products of the MD and GED (Supp. Fig. 2). We solved the phase problem by a single anomalous dispersion protocol and could build and refine a model containing two molecules in the asymmetric unit (Methods, Supp. Table 1 and 2). Each monomer spans nearly the complete MD and the amino(N-)-terminal part of the GED (amino acids 366-633) which together fold into an elongated anti-parallel fourhelical bundle where the MD contributes three helices and the GED one (Fig. 1b, Supp. Fig. 3). This segment corresponds to the stalk region of dynamin 7 , and we refer to it as stalk of MxA. The first visible amino acid, Glu366, is 15 amino acids downstream of the last visible residue of the corresponding G-domain structure in rat dynamin (Supp. Fig. 3) 8 . It marks the start of helix α1 in the MxA stalk which is divided in α1 N and α1 C by a 10 amino acid long loop, L1, introducing a 30° kink. A putative loop L2 (amino acids 438-447) opposite of the deduced position of the G-domain is not visible in our structure. L2 was previously demonstrated to be the target of a functionally neutralising monoclonal antibody 9,10 . Helix α2 runs anti-parallel to α1 back to the G-domain. It ends in a short loop L3 and is followed by helix α3 that extends in parallel to α1. The 40 amino acid long loop L4 (residues 532-572) is at the equivalent sequence position as the PH domain of dynamin (Fig. 1a, Supp. Fig. 3) and is absent in our model. L4 is predicted to be unstructured and was previously shown to be proteinase K sensitive 11 .At the C-terminus, the GED supplies 44 residues to helix α4 which proceeds in parallel to helix α2 back to the G-domain. It is followed by a short helix α5 which directs the polypeptide chain towards the N-terminus of the MD. The carboxy(C-)-terminal 30 highly conserved residues of the GED known to be involved in antiviral specificity 12 are missing in our model. In dynamin, the corresponding residues were shown to directly interact with the G-domain 13 . The stalk of MxA is divergent from the corresponding structures of other dynamin superfamily members, such as GBP1 14 , EHD2 15 and BDLP 16 although some features are shared (Supp. Fig. 4). 4 In the crystal lattice, each MxA stalk is assembled in a criss-cross pattern resulting in a linear oligomer, where each stalk contributes three distinct interfaces (Fig. 1c). Such an arrangement of the stalks is plausible for the Mx oligomer since all G-domains would be located at one side of the oligomer whereas the putative substrate-binding site in L2 and L4 would be located at the opposite side (Fig. 1b, c).The hydrophobic interface-1 covering 1300 Å 2 is conserved among Mx proteins and dynamins and has a two-fold symmetry between the associating monomers (Fig....

Human myxovirus resistance protein 1 (MxA) is an interferon-induced dynamin-like GTPase that acts as a cell-autonomous host restriction factor against many viral pathogens including influenza viruses. To study the molecular principles of its antiviral activity, we determined the crystal structure of nucleotide-free MxA, which showed an extended three-domain architecture. The central bundle signaling element (BSE) connected the amino-terminal GTPase domain with the stalk via two hinge regions. MxA oligomerized in the crystal via the stalk and the BSE, which in turn interacted with the stalk of the neighboring monomer. We demonstrated that the intra- and intermolecular domain interplay between the BSE and stalk was essential for oligomerization and the antiviral function of MxA. Based on these results, we propose a structural model for the mechano-chemical coupling in ring-like MxA oligomers as the principle mechanism for this unique antiviral effector protein.

SummaryInterferon exposure boosts cell-autonomous immunity for more efficient pathogen control. But how interferon-enhanced immunity protects the cytosol against bacteria and how professionally cytosol-dwelling bacteria avoid clearance are insufficiently understood. Here we demonstrate that the interferon-induced GTPase family of guanylate-binding proteins (GBPs) coats Shigella flexneri in a hierarchical manner reliant on GBP1. GBPs inhibit actin-dependent motility and cell-to-cell spread of bacteria but are antagonized by IpaH9.8, a bacterial ubiquitin ligase secreted into the host cytosol. IpaH9.8 ubiquitylates GBP1, GBP2, and GBP4 to cause the proteasome-dependent destruction of existing GBP coats. This ubiquitin coating of Shigella favors the pathogen as it liberates bacteria from GBP encapsulation to resume actin-mediated motility and cell-to-cell spread. We conclude that an important function of GBP recruitment to S. flexneri is to prevent the spread of infection to neighboring cells while IpaH9.8 helps bacterial propagation by counteracting GBP-dependent cell-autonomous immunity.

Cell-autonomous immunity relies on the ubiquitin coat surrounding cytosol-invading bacteria functioning as an 'eat-me' signal for xenophagy. The origin, composition, and precise mode of action of the ubiquitin coat remain incompletely understood. Here, by studying Salmonella Typhimurium, we show the E3 ligase LUBAC to generate linear (M1-linked) polyubiquitin patches in the ubiquitin coat that serve as anti-bacterial and pro-inflammatory signaling platforms. LUBAC is recruited via its subunit HOIP to bacterial surfaces that are no longer shielded by host membranes and already displaying ubiquitin, suggesting LUBAC amplifies and refashions the ubiquitin coat. LUBAC-synthesized polyubiquitin recruits Optineurin and Nemo for xenophagy and local activation of NF-κB, respectively, which independently restrict bacterial proliferation. In contrast, the professional cytosol-dwelling Shigella flexneri escapes from LUBAC-mediated restriction through the antagonizing effects of the effector E3 ligase IpaH1.4 on the deposition of M1-linked poly-ubiquitin and the subsequent recruitment of Nemo and Optineurin. We conclude that LUBAC-synthesized M1-linked ubiquitin transforms bacterial surfaces into signaling platforms for anti-bacterial immunity reminiscent of anti-viral assemblies on mitochondria.Mammalian cells maintain a sterile cytosol by deploying galectins to survey endomembrane damage and subsequently coat invading bacteria as well as damaged membranes with polyubiquitin 1. The bacterial ubiquitin coat comprises multiple linkage types, synthesized by several E3 ligases such as LRSAM1, Parkin, Smurf1 and RNF166 2-5. Galectin-8 and the poly-ubiquitin coat provide ligands ('eat-me' signals) for NDP52, Optineurin and other cargo receptors that induce anti-bacterial autophagy ('xenophagy') and restrict bacterial proliferation 1,6-10.

Balanced fusion and fission are key for proper function and physiology of mitochondria 1,2 . Remodelling of the mitochondrial inner membrane (IM) is mediated by dynamin-like Mitochondrial genome maintenance 1 protein (Mgm1) in fungi or the related Optic atrophy protein 1 (OPA1) in animals [3][4][5] . Mgm1 is required for the preservation of mitochondrial DNA in yeast 6 , whereas mutations in the OPA1 gene in humans are a common cause for autosomal dominant optic atrophy, a genetic disorder affecting the optical nerve 7,8 . Mgm1 and OPA1 are present in mitochondria as a membrane-integral long (l) form and a short (s) form that is soluble in the intermembrane space. Yeast strains expressing temperaturesensitive mutants of Mgm1 9,10 or mammalian cells devoid of OPA1 display fragmented mitochondria 11,12 , suggesting an important role of Mgm1/OPA1 in IM fusion. Consistently, only the mitochondrial outer membrane (OM), but not the IM, fuses in the absence of functional Mgm1 13,14 . Mgm1 and OPA1 have also been shown to maintain proper cristae architecture 10,14 . For example, OPA1 prevents the release of pro-apoptotic factors by tightening cristae junctions 15 . Finally, s-OPA1 localises to mitochondrial constriction sites, where it presumably promotes mitochondrial fission 16 . How Mgm1/OPA1 perform their diverse functions in membrane fusion, scission, and cristae organisation is at present unknown. Here, we present crystal and electron cryo-tomography (cryo-ET) structures of Chaetomium thermophilum Mgm1. Mgm1 consists of a GTPase domain, a bundle signalling element (BSE) domain, a stalk, and a paddle domain containing a membrane binding site. Biochemical and cell-based experiments demonstrate that the Mgm1 stalk mediates assembly of bent tetramers into helical filaments. Cryo-ET of Mgm1-decorated lipid tubes and fluorescence microscopy experiments on reconstituted membrane tubes indicate how the tetramers assemble on positively or negatively curved membranes. Our findings convey how Mgm1/OPA1 filaments dynamically remodel the mitochondrial IM.We purified and crystallised a truncated s-Mgm1 isoform from the thermophilic fungus Chaetomium thermophilum (from here on Mgm1) (Fig. 1a, Extended Data Fig. 1a, Supplementary Data Fig. 1). Crystals of this construct grown in the absence of nucleotides diffracted to 3.6 Å resolution. The structure was solved by single anomalous dispersion (Extended Data Fig. 1b, c, Extended Data Table 1).The structure of Mgm1 contains four domains: A G domain, a bundle signalling element (BSE) domain, a stalk, and a paddle (Fig. 1a, b). The G domain closely resembles that of human dynamin (Extended Data Fig. 2). An interface across the nucleotide-binding site responsible for G domain dimerisation in the dynamin superfamily (the 'G interface') is highly conserved in Mgm1 (Extended Data Fig. 1e). The adjacent BSE domain consists of three helices derived from different regions of Mgm1 (Fig. 1a, b). The BSE domain contacts the G domain, as in the closed conformation of dynamin [17][18][19] . The M...